Use of compound in preparation of drug for preventing, relieving, assisting in treating, or treating lung injury

Abstract

A use of a compound represented by formula I, or a cis-trans isomer, enantiomer, diastereomer, racemate, solvate, hydrate, pharmaceutically acceptable salt or prodrug thereof in the preparation of a drug for preventing, relieving, assisting in treating, or treating lung injury. Upon verification, administration of the compound represented by formula I can significantly reduce proinflammatory cytokines, i.e., tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6), of a rat model of acute lung injury, reduce the infiltration of inflammatory cells (comprising monocytes / macrophages, lymphocytes, and neutrophils) in lung tissues, relieve pathological injury of the lung tissues, improve the lung function, and increase the survival rate of comorbidities of acute lung injury. The compound represented by formula I can be used for preventing, relieving, assisting in treating, or treating lung injury, especially acute lung injury or comorbidities thereof, or acute respiratory distress syndrome or comorbidities thereof.

Description

Use of a compound in preparing a drug for preventing, alleviating, assisting in the treatment or treating lung injury

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application filed with the China Patent Office on June 29, 2023, with application number 202310787795.4, and invention name “Use of a compound in the preparation of a drug for preventing, alleviating, assisting in the treatment or treating lung injury”, the entire contents of which are incorporated herein by reference; this application also claims priority to the Chinese patent application filed with the China Patent Office on June 28, 2024, with application number 202410867721.6, and invention name “Use of a compound in the preparation of a drug for preventing, alleviating, assisting in the treatment or treating lung injury”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to the field of medicine, and specifically to the use of a compound in the preparation of a drug for preventing, alleviating, assisting in the treatment or treating lung injury. Background Art

[0004] Novel coronavirus pneumonia (COVID-19) is a serious illness caused by the novel coronavirus 2 (SARS-CoV-2). COVID-19 patients often experience acute respiratory distress syndrome (ARDS), which can lead to severe acute respiratory failure and extremely high mortality. Patients with chronic conditions such as diabetes or cardiovascular disease are more likely to develop severe illness or even death after SARS-CoV-2 infection.

[0005] Studies have shown that excessive production of proinflammatory cytokines is a major factor in the development of ARDS. Therefore, blocking the production of proinflammatory cytokines may help prevent the progression of ARDS in COVID-19 patients. Furthermore, studies have found that autopsies of lung tissue from fatal COVID-19 patients show strong macrophage infiltration, suggesting that controlling the excessive inflammatory response produced by macrophages may have a beneficial effect on the development of ARDS in COVID-19 patients.

[0006] Summary of the Invention

[0007] Therefore, the technical problem to be solved by the present application is the use of a compound in the preparation of a drug for preventing, alleviating, assisting in the treatment or treating lung injury.

[0008] To this end, this application provides the following technical solutions:

[0009] Use of the compound represented by Formula I, its cis-trans isomers, enantiomers, diastereomers, racemates, solvates, hydrates, or pharmaceutically acceptable salts or prodrugs thereof in the preparation of a medicament for preventing, alleviating, assisting in the treatment, or treating lung injury;

[0010] R is H, fluoro or cyano;

[0011] X is N or CR1, and R1 is H.

[0012] Optionally, the compound represented by formula I is:

[0013] The use of the compound represented by formula I, its cis-trans isomers, its enantiomers, its diastereomers, its racemates, its solvates, its hydrates or its pharmaceutically acceptable salts or their prodrugs in combination with ceftazidime in the preparation of a drug for preventing, alleviating, assisting in the treatment or treating lung injury, or in preventing, alleviating, assisting in the treatment or treating lung injury.

[0014] Optionally, the lung injury includes: acute lung injury or its combined diseases; or

[0015] Acute respiratory distress syndrome or its complications.

[0016] Optionally, the combined disease is diabetes;

[0017] Optionally, the diabetes is type 1 diabetes.

[0018] Optionally, the lung injury is caused by infection, pro-inflammatory cytokine storm and / or inflammatory cell infiltration.

[0019] Optionally, the infection comprises a microbial infection;

[0020] Optionally, the microbial infection includes viral infection or bacterial infection;

[0021] Optionally, the viral infection is a coronavirus infection;

[0022] Optionally, the coronavirus infection is SARS-CoV-2 virus infection.

[0023] Optionally, the proinflammatory cytokines in the proinflammatory cytokine storm are TNF-α and / or IL-6.

[0024] Optionally, the inflammatory cell infiltration is monocyte / macrophage, lymphocyte and neutrophil infiltration;

[0025] Optionally, the macrophage infiltration is an increased proportion of CD68 positive cells.

[0026] Optional, including the following uses:

[0027] (1) Use in the preparation of drugs for inhibiting pro-inflammatory cytokine storm;

[0028] (2) Use in the preparation of drugs for inhibiting inflammatory cell infiltration.

[0029] A pharmaceutical preparation for preventing, alleviating, assisting in the treatment or curing lung injury, comprising:

[0030] The active ingredient is a compound represented by formula I, its cis-trans isomers, its enantiomers, its diastereomers, its racemates, its solvates, its hydrates, or its pharmaceutically acceptable salts or prodrugs thereof; and

[0031] Pharmaceutically acceptable pharmaceutical excipients or carriers.

[0032] Optionally, the active ingredient further comprises ceftazidime;

[0033] Optionally, the pharmaceutical preparation for preventing, alleviating, assisting in the treatment or treating lung injury includes liquid preparations and solid preparations.

[0034] A method for preventing, alleviating, adjuvant therapy or treating lung injury, characterized by administering to a subject in need thereof a compound represented by Formula I, its cis-trans isomers, its enantiomers, its diastereomers, its racemates, its solvates, its hydrates, its pharmaceutically acceptable salts or its prodrugs;

[0035] R is H, fluoro or cyano;

[0036] X is N or CR1, and R1 is H.

[0037] Optionally, the compound represented by formula I is:

[0038] Optionally, the compound represented by Formula I, its cis-trans isomers, its enantiomers, its diastereomers, its racemates, its solvates, its hydrates, its pharmaceutically acceptable salts or its prodrugs are used in combination with ceftazidime.

[0039] Optionally, the lung injury includes: acute lung injury or its combined diseases; or acute respiratory distress syndrome or its combined diseases; or chronic obstructive pulmonary disease or its combined diseases.

[0040] Optionally, the combined disease is diabetes;

[0041] Optionally, the diabetes is type 1 diabetes.

[0042] Optionally, the lung injury is caused by infection, pro-inflammatory cytokine storm and / or inflammatory cell infiltration.

[0043] Optionally, the infection comprises a microbial infection;

[0044] Optionally, the microbial infection includes viral infection or bacterial infection;

[0045] Optionally, the viral infection is a coronavirus infection;

[0046] Optionally, the coronavirus infection is SARS-CoV-2 virus infection.

[0047] Optionally, the proinflammatory cytokines in the proinflammatory cytokine storm are TNF-α and / or IL-6.

[0048] Optionally, the inflammatory cell infiltration is monocyte / macrophage, lymphocyte and neutrophil infiltration;

[0049] Optionally, the macrophage infiltration is an increased proportion of CD68 positive cells.

[0050] The technical solution of this application has the following advantages:

[0051] 1. The present application provides a compound of Formula I, its cis-trans isomers, enantiomers, diastereomers, racemates, solvates, hydrates, or pharmaceutically acceptable salts or prodrugs thereof, for use in the preparation of a medicament for preventing, alleviating, adjuvant therapy, or treating lung injury. This application has demonstrated that administration of the compound of Formula I can significantly reduce proinflammatory cytokines (tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6)) in a rat model of acute lung injury, inhibit lipoprotein-associated phospholipase A2 (Lp-PLA2) activity, improve the infiltration of monocytes / macrophages, lymphocytes, and neutrophils in lung tissue, improve lung tissue pathological damage, improve lung function, and increase the survival rate of patients with acute lung injury and a concurrent disease (diabetes). The above indicates that the compound of Formula I can be used to prevent, alleviate, adjuvant therapy, or treat lung injury, particularly acute lung injury or its concurrent disease (diabetes) or acute respiratory distress syndrome or its concurrent disease (diabetes). BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific implementation methods or the description of the prior art. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0053] FIG1 is an experimental flow chart of Experimental Example 1 of the present application;

[0054] Figure 2 shows the Lp-PLA2 activity in the plasma of rats in different treatment groups in Experimental Example 1 of the present application. All experimental data are expressed as mean ± standard error. Compared with vehicle, * represents p < 0.05, *** represents p < 0.001;

[0055] Figure 3 shows the results of cytokine determination in rat BALF in Experiment 1 of this application. All experimental data are expressed as mean ± standard error. Compared with vehicle, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.

[0056] FIG4 is an experimental flow chart of Experimental Example 2 of this application;

[0057] FIG5 shows the effect of SR19 on the survival rate of diabetic ALI rats induced by different doses of LPS in Experimental Example 2 of the present application; V in the figure represents Vehicle, and SR represents SR19;

[0058] Figure 6 shows the Lp-PLA2 activity in the plasma of SD rats in different treatment groups in Experimental Example 2 of the present application. All experimental data are expressed as mean ± standard error, * represents p < 0.05, *** represents p < 0.001;

[0059] Figure 7 shows the results of macrophage infiltration assay in rat lung tissue. All experimental data are expressed as mean ± standard error. In the figure, *** and **** represent p < 0.001 and p < 0.0001, respectively.

[0060] FIG8 is an experimental flow chart of Experimental Example 3;

[0061] Figure 9 shows the inhibitory effect of SR15 on Lp-PLA2 enzyme activity in the plasma of mice with PA-induced acute pneumonia in Experimental Example 3; all experimental data are expressed as mean ± standard error, and one-way ANOVA was performed using Dunnett's method for multiple comparisons. Compared with vehicle, * represents p < 0.05, *** represents p < 0.001, and "ns" indicates: not statistically significant.

[0062] Figure 10 shows chemokine levels and inflammatory cell levels in BALF in Experimental Example 3. All experimental data are expressed as mean ± standard error. Ordinary one-way ANOVA was performed using Dunnett's method for multiple comparisons. Compared with vehicle, * represents p < 0.05, and *** represents p < 0.001.

[0063] Figure 11 Pathological damage scores of mouse lung tissue in Experimental Example 3; All experimental data are expressed as mean ± standard error; Improvement rate calculation formula: Improvement rate (%) = (score vehicle -Rating SR15 or CAZ or SR15 / CAZ) / Rating vehicle *100 or improvement rate (%) = (score CAZ -score SR15 / CAZ ) / score vehicle *100;

[0064] FIG12 is a flowchart of the experiment in Experimental Example 4;

[0065] FIG13 shows the levels of Lp-PLA2 and oxLDL in the plasma of rats in each group in Experimental Example 4. All experimental data are expressed as mean ± standard error. Ordinary one-way ANOVA was performed with Dunnett's method for multiple comparisons. Compared with Day 0, * represents p < 0.05, and *** represents p < 0.001.

[0066] FIG14 shows the results of monocyte counts in BALF in Experimental Example 4; all experimental data are expressed as mean ± standard error, inhibition rate (%) = (count [vehicle] - count [SR19]) / count [vehicle] * 100;

[0067] Figure 15 shows the LysoPC detection levels in BALF in Experimental Example 4; all experimental data are expressed as mean ± standard error, inhibition rate (%) = (counts [vehicle] - counts [SR19]) / counts [vehicle] * 100; Dunnett's method was used for multiple comparisons, compared with "Vehicle", * represents p < 0.05, # < 0.01. Note: The control sample is the test sample added when testing LysoPC;

[0068] Figure 16 Analysis of lung function PenH index; all experimental data are expressed as mean ± standard error, "t test" analysis, compared with vehicle, ** represents p < 0.01.

[0069] FIG17 is a flow chart of the experimental design of time-dependent drug efficacy evaluation in Experimental Example 5.

[0070] FIG18 is a flow chart of the experimental design for dose-dependent efficacy evaluation in Experimental Example 5.

[0071] Figure 19 shows the Lp-PLA2 activity in the plasma of rats in different treatment groups in Experimental Example 5; all experimental data are expressed as mean ± standard error, compared with STZ / LPS+Vehicle, *, # represent p < 0.05, ## represents p < 0.01, *** represents p < 0.001.

[0072] FIG20 shows the white blood cell counts in BALF of rats in Experimental Example 5; all experimental data are expressed as mean ± standard error, and * represents p < 0.05 compared with STZ / LPS vehicle.

[0073] FIG21 shows the differential counts of monocytes, lymphocytes and neutrophils in BALF of rats in Experimental Example 5; all experimental data are expressed as mean ± standard error, compared with STZ / LPS+Vehicle.

[0074] Figure 22 shows the albumin level in BALF of rats in Experimental Example 5; all experimental data are expressed as mean ± standard error, compared with STZ / LPS+Vehicle, * represents p < 0.05, ns represents no significance.

[0075] Figure 23 shows the pathological analysis of lung tissue stained with HE staining in Experimental Example 5. All experimental data are presented as mean ± standard error. Ordinary one-way ANOVA was used for multiple comparisons. * indicates p < 0.05, ** indicates p < 0.01, and ns indicates no significance compared with vehicle. The treatment group received 10 mg / kg SR13. DETAILED DESCRIPTION

[0076] The following examples are provided to further better understand the present application, but are not limited to the best implementation mode described herein, and do not limit the content and protection scope of the present application. Any product identical or similar to the present application obtained by anyone under the inspiration of the present application or by combining the features of the present application with other prior arts shall fall within the scope of protection of the present application.

[0077] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0078] The following compounds:

[0079] The compounds represented by the above formula I(1), formula I(2) and formula I(3) are known compounds and are prepared by Examples 13, 19 and 15 of patent document WO2021089032, respectively. In the following examples, the compound represented by formula I(1) is numbered SR13, the compound represented by formula I(3) is numbered SR19, and the compound represented by formula I(3) is numbered SR15.

[0080] Experimental Example 1: Efficacy of SR13 on LPS intratracheal instillation-induced acute lung injury model in rats

[0081] 1. Experimental purpose: Studies have shown that the LPS-induced acute lung injury (ALI) model in rats can simulate the cytokine signaling pathways and lung tissue damage that occur in ARDS in COVID-19 patients (GUAN WJ, NI ZY, HU Y, et al. Clinical Characteristics of Coronavirus Disease 2019 in China[J]. N Engl J Med, 2020, 382(18): 1708-1720). Therefore, in this example, an LPS tracheal instillation-induced acute lung injury model in rats was constructed to investigate the efficacy of SR13 in acute lung injury and ARDS.

[0082] 2. Experimental design

[0083] 1) SR13 efficacy in animals

[0084] Adult healthy male Sprague-Dawley rats (280-330 g, 6-8 weeks old) were instilled with lipopolysaccharide (LPS) via tracheal instillation to induce an acute lung injury (ALI) rat model. The ALI rats were randomly divided into three treatment groups (SR13 doses of 1 mg / kg, 3 mg / kg, and 10 mg / kg) and a vehicle group. Sham-operated rats of the same age served as the sham control group. SR13 and vehicle were administered by single oral gavage daily for two consecutive days. The control group received an equal volume of saline via tracheal instillation (see Table 1 for details). On the second day, after administration, LPS was instilled into the trachea. Six hours after surgery, plasma and bronchoalveolar lavage fluid (BALF) were collected. Plasma Lp-PLA2 activity was measured using an enzymatic assay using thio-PAF as a substrate, and BALF cytokine levels were measured using enzyme-linked immunosorbent assay. The experimental process is shown in Figure 1.

[0085] 2) SR13 inhibits the in vitro activity of Lp-PLA2 in the plasma of healthy and ALI rats

[0086] Plasma from healthy and ALI rats was collected, and compound solutions with 10 consecutive compound concentrations were prepared to detect the inhibitory effects of different concentrations of compounds on Lp-PLA2 activity in the plasma of healthy and ALI rats.

[0087] Table 1. Animal groups and dosing regimens for the in vivo efficacy experiment of SR13

[0088] Note: PO is oral administration; QD is once a day.

[0089] 3. Experimental Preparation

[0090] 3.1 Test product information

[0091] Table 2

[0092] 3.2 Reagent Information

[0093] Table 3

[0094] 3.3 Main instruments and equipment

[0095] Table 4

[0096] 3.4 Animals and breeding environment

[0097] This experiment used male adult Sprague-Dawley rats, weighing 280–330 g and aged 6–8 weeks, provided by Zhejiang Weitong Lihua Laboratory Animal Co., Ltd. Animals were housed in standard polypropylene cages at a stable temperature of 22–24°C under a 12-h light / dark cycle. Animals had free access to water and food.

[0098] 3.5 SR13 in vivo efficacy in animals: experimental preparations

[0099] Solvent: Weigh 10 g of MC powder and dilute to 1 L with deionized water to prepare a 1% MC solution. Vortex and stir at room temperature overnight and store in a refrigerator at 4°C until use.

[0100] LPS preparation: Weigh an appropriate amount of LPS powder and dissolve it in normal saline to prepare a solution with a concentration of 2 mg / mL. Vortex and sonicate until a clear solution is formed.

[0101] SR13 preparation: Weigh an appropriate amount of compound SR13 powder and add 1% MC solution according to the proportion. Vortex and sonicate until a uniform suspension is formed.

[0102] 3.6 In vitro inhibition of Lp-PLA2 activity by SR13 in plasma of healthy and ALI rats: experimental preparations

[0103] Preparation of experimental working solution:

[0104] 1× Assay buffer: 100 mM Tris-base, 1 mM EGTA (adjust pH to 7.2 with HCl);

[0105] 2×Substrate solution: 0.5 mM 2-thio-PAF;

[0106] 10× DTNB solution: 10 mM DTNB.

[0107] Compound solution preparation:

[0108] SR13 Solution: Prepare SR13 to a 10 mM stock solution in DMSO. Serially dilute 3-fold with DMSO to obtain 10 consecutive compound concentrations. Each concentration point is then diluted 50-fold with 1× Assay buffer to obtain working solutions of the compound at concentrations of 200, 66.67, 22.22, 7.41, 2.47, 0.82, 0.27, 0.09, 0.03, and 0.01 μM. The final concentrations of the compound after addition to the reaction system are: 10, 3.33, 1.11, 0.37, 0.12, 0.04, 0.01, 0.005, 0.002, and 0.0005 μM.

[0109] 4. Experimental methods and steps

[0110] 4.1 Efficacy of SR13 in Animals

[0111] 4.1.1 Establishment of rat model of acute lung injury

[0112] After anesthesia, the rat's limbs were secured to a surgical board. The neck was shaved and disinfected with iodine. An approximately 1-cm incision was made in the neck, and the muscles were bluntly dissected to expose the trachea. LPS or saline was slowly and evenly infused into the trachea over 30 seconds using a 1-mL syringe with a 27-gauge needle. Immediately after injection, the rat was placed upright with its head up and its tail down for 1 minute to allow for adequate fluid flow. The wound was then sutured. The rats were placed on a heating pad to maintain body temperature until they regained consciousness.

[0113] 4.1.2 Dosage Information

[0114] The dosing volume for each rat was based on its specific body weight. The standard dosing volume for SR13 was 10 mL / kg, and the standard dosing volume for LPS was 1 mL / kg. The actual animal doses for 0.1 mg / mL, 0.3 mg / mL, and 1 mg / mL SR13 solutions were 1 mg / kg, 3 mg / kg, and 10 mg / kg, respectively. The LPS dose was 2 mg / kg. The dosing volume for each rat was based on its body weight and the concentrations of the prepared SR13 suspension and LPS solution. The dosing volume calculation formula is: Dosing volume (mL) = body weight (kg) × (Dosage (mg / kg) / (SR13 or LPS) concentration (mg / mL)).

[0115] 4.1.3 Sample collection

[0116] 4.1.3.1 Blood sample collection

[0117] All animals underwent whole blood sample collection 6 hours after LPS modeling. The blood samples were collected in centrifuge tubes containing K2EDTA, centrifuged at 5000 rpm for 10 minutes (4°C) to separate the plasma, and then stored in a -80°C refrigerator for analysis.

[0118] 4.1.3.2 BALF sample collection

[0119] At the end of the experiment, the animals were anesthetized and sacrificed by cardiac exsanguination. The thoracic cavity and cervical trachea were surgically exposed. The right lung was ligated, and the left lung was lavaged twice with PBS via endotracheal intubation, with each lavage repeated three times. BALF was collected. BALF with a recovery rate of 80% or more was used for subsequent analysis.

[0120] 4.1.2 Sample determination

[0121] 4.1.4.1 Methodology for testing Lp-PLA2 activity in plasma

[0122] The assay for measuring Lp-PLA2 activity in plasma and the inhibition of Lp-PLA2 activity by SR13 in healthy and ALI rat plasma were performed using 2-thio-PAF as the substrate. Kinetic analysis was performed using a multifunctional microplate reader with absorbance readings at 405 nm and 510 nm. Lp-PLA2 activity was expressed as the amount of substrate hydrolyzed per milliliter of plasma per unit time (nmol / min / mL).

[0123] The specific protocol for testing Lp-PLA2 activity in plasma obtained from the in vivo pharmacodynamics experiment is as follows: thaw the plasma at room temperature, level it and place it in a centrifuge at 2000 rpm for 1 minute; add 20 μL of plasma to a 96-well assay plate (brand: Greiner, catalog number: 655101), add 15 μL of 1× assay buffer to each well, and oscillate to mix thoroughly for use; negative control well (LC): according to the 96-well assay plate, add 5 μL of 200 μM Darapladib solution (positive compound, compound diluted with 1× assay buffer) to each well; for other experimental wells, according to the 96-well assay plate, add 5 μL of 2% DMSO solution (diluted with 1× assay buffer) to each well; add 10 μL of 10× DTNB suspension to the assay plate, oscillate to mix until the solution is clear, and incubate at room temperature for 30 minutes; after incubation, place in a 37°C biochemical incubator for 5 to 10 minutes, add 50 μL The reaction was initiated by adding 2× substrate solution to the test plate. The test plate was shaken and mixed, and placed in a microplate reader with the temperature set to 37°C. Kinetic detection was performed at a wavelength of 405 / 510 nm.

[0124] 4.1.4.2 BALF Cytokine Analysis: ELISA was used to measure TNF-α and IL-6 cytokine levels in BALF using a multifunctional microplate reader at wavelengths of 450 nm and 570 nm.

[0125] 4.2 The specific protocol for the in vitro inhibition test of SR13 on Lp-PLA2 in the plasma of healthy and ALI rats is as follows:

[0126] 1) Thaw plasma from healthy rats and ALI rats at room temperature, balance the plasma, and centrifuge at 2000 rpm for 1 minute.

[0127] 2) Add 20 μL of plasma to a 96-well assay plate (Greiner, Cat. No. 655101), add 15 μL of 1× assay buffer to each well, and vortex to mix.

[0128] 3) The compound was serially diluted 3-fold in DMSO solution to 10 concentration points;

[0129] 4) The diluted compound was diluted 50-fold with 1× Assay buffer and 5 μL of compound dilution was added to each well of the 96-well assay plate;

[0130] Positive control wells (HC): Add 5 μL of 2% DMSO solution (diluted with 1× assay buffer) to the 96-well assay plate according to the plate layout;

[0131] Negative control wells (LC): Add 5 μL of 200 μM Darapladib solution (positive compound, diluted with 1× Assay buffer) to the 96-well assay plate according to the plate layout;

[0132] 5) Add 10 μL of 10× DTNB suspension to the assay plate, shake until the solution is clear, and incubate at room temperature for 30 minutes;

[0133] 6) After incubation, place the plate in a 37°C biochemical incubator for 5-10 minutes. Add 50 μL of 2× substrate solution to the assay plate to initiate the reaction.

[0134] 7) The assay plate was shaken and mixed, and placed in a microplate reader with the temperature set to 37°C. Kinetic assays were performed at wavelengths of 405 and 510 nm.

[0135] 4.3 Statistical analysis

[0136] 4.3.1 SR13 efficacy in animals:

[0137] 4.3.1.1 Analysis of Lp-PLA2 activity in plasma. Experimental data were collected using Biotek Synergy H1, Software Version Gen 5, and analyzed using Microsoft Excel 2010. One-way ANOVA and Dunnett's multiple comparisons test were used to analyze the differences between the groups. Statistical results are expressed as mean ± standard error. p < 0.05 indicates a statistically significant difference. SR13 inhibits Lp-PLA2 activity in the plasma of healthy and ALI rats. IC 50 The values ​​were analyzed by nonlinear regression using the Software Version Gen 5 software provided with the Biotek microplate reader.

[0138] 4.3.1.2 BALF cytokine analysis was performed using Microsoft Excel 2010. Differences between groups were analyzed using one-way ANOVA and Dunnett's multiple comparisons test. Statistical results are presented as mean ± standard error. A p < 0.05 indicated statistical significance.

[0139] 4.3.2 Inhibition of Lp-PLA2 Activity in the Plasma of Healthy and ALI Rats by SR13 in Vitro

[0140] In the experiment, experimental control conditions were defined based on the signal. The average reaction rate of the Lp-PLA2 enzyme in the wells containing plasma and 0.1% DMSO was used as the positive control for no inhibition (defined as "High control"); the average reaction rate of the Lp-PLA2 enzyme in the wells containing plasma and 10 μM Darapladib was used as the negative control for 100% inhibition (defined as "Low control"). The inhibition rate was calculated using the following formula: Inhibition rate (%) = [1-(experimental wells – average value)] [阴性控制] ) / (average value [阳性控制] -average value [阴性控制] )] × 100, and data are expressed as mean ± standard deviation. Nonlinear regression analysis was performed using Software Version Gen 5 software.

[0141] 5 Experimental results and discussion

[0142] 5.1 SR13 efficacy in animals:

[0143] 5.1.1 Results of plasma Lp-PLA2 activity determination

[0144] Plasma Lp-PLA2 activity testing showed significant increases in plasma Lp-PLA2 activity in ALI rats (p < 0.05) (Figure 2). Compared to the vehicle group, plasma Lp-PLA2 activity decreased in the 1, 3, and 10 mg / kg / day SR13 groups by 76.39 ± 1.98% (mean ± standard error, n = 9), 91.19 ± 1.38% (mean ± standard error, n = 9), and 99.47 ± 0.38% (mean ± standard error, n = 9), respectively, in a dose-dependent manner. The results are shown in Table 5 and Figure 2, where mpk = mg / kg.

[0145] Table 5. SR13 inhibits Lp-PLA2 activity in the plasma of ALI rats

[0146] 5.1.2 Cytokine Test Results in BALF

[0147] In the ALI rat model, 6 hours after LPS stimulation, BALF TNF-α and IL-6 levels were significantly elevated (p < 0.001) (Figure 3). Compared with the vehicle, BALF TNF-α levels decreased by 28.93 ± 12.24% (mean ± standard error, n = 14), 34.03 ± 10.37% (mean ± standard error, n = 14), and 47.23 ± 6.82% (mean ± standard error, n = 14) in the 1 mg / kg, 3 mg / kg, and 10 mg / kg SR13 groups, respectively. BALF IL-6 levels decreased by 20.03 ± 19.95% (mean ± standard error, n = 14), 7.49 ± 15.89% (mean ± standard error, n = 14), and 54.91 ± 5.61% (mean ± standard error, n = 14), respectively. SR13 inhibited the elevation of BALF cytokines in a dose-dependent manner. (Table 6, Figure 3)

[0148] Table 6. SR13 inhibits the increase of cytokines in BALF

[0149] In summary, 6 hours after LPS induction, ALI rats in the vehicle group (LPS+vehicle) showed significant increases in plasma Lp-PLA2 activity (p < 0.05) and TNF-α and IL-6 levels in bronchoalveolar lavage fluid (BALF) (p < 0.001). SR13 had a dose-dependent inhibitory effect on the elevation of plasma Lp-PLA2 activity and BALF TNF-α and IL-6 levels.

[0150] 5.2 In vitro inhibition of Lp-PLA2 activity by SR13 in plasma of healthy and ALI rats

[0151] As shown in Tables 7 and 8 below, SR13 inhibits Lp-PLA2 activity in the plasma of healthy and ALI rats. 50 They were 45.94±8.54 nM (mean±standard deviation, n=3) and 31.92 nM (mean, n=2), respectively.

[0152] Table 7. Summary of the inhibition data of SR13 on Lp-PLA2 activity in plasma of healthy SD rats

[0153] Table 8. Summary of the inhibition data of SR13 on Lp-PLA2 activity in the plasma of ALI rats

[0154] Experimental Example 2: Efficacy of SR19 on LPS-induced acute lung injury model in rats

[0155] 1. Objective: Previous studies have shown that the rat model of acute lung injury (ALI) induced by intratracheal instillation of LPS can mimic cytokine signaling pathways and lung tissue damage similar to ARDS in COVID-19 patients. This study utilized rats with type 1 diabetes combined with ALI to simulate COVID-19-related ARDS in the presence of diabetes. This study aimed to evaluate the efficacy of SR19 in the rat model of acute lung injury (ALI) induced by intratracheal instillation of LPS in type 1 diabetic rats.

[0156] 2. Experimental design

[0157] 1) SR19 efficacy in animals

[0158] After the rats arrived at the facility and acclimatized for 2-3 days, they were intraperitoneally injected with STZ solution (60 mg / kg) to induce type 1 diabetes. One week after STZ injection, rats with blood glucose levels greater than 16.7 mmol / L were screened for LPS-induced acute lung injury modeling.

[0159] Animals were divided into seven experimental groups: Naive (normal rats), Naive + Vehicle, Naive + SR19, LPS + Vehicle, LPS + SR19, STZ + LPS + Vehicle, and STZ + LPS + SR19. The SR19 dose was 10 mg / kg. SR19 and vehicle were administered daily by gavage for three consecutive days, starting one day before LPS induction (see Figure 4). The rats were observed after LPS induction and mortality was recorded. At the end of the experiment, 48 hours after LPS induction, lung tissue and plasma were collected for subsequent analysis.

[0160] The lung tissue macrophage infiltration test is a single-blind test. After sampling the animal lung tissue, the lung tissue sample with only the animal number information will be handed over to Nantong WuXi AppTec Pharmaceutical Technology Co., Ltd. for IHC staining analysis. After Nantong WuXi AppTec Pharmaceutical Technology Co., Ltd. has fed back all the original data, the test will be unblinded for data analysis.

[0161] 2) SR19 inhibits the in vitro activity of Lp-PLA2 in the plasma of healthy SD rats

[0162] Plasma of healthy SD rats was obtained, and compound solutions with 10 consecutive compound concentrations were prepared to detect the inhibitory effects of compounds at different concentrations on Lp-PLA2 activity in the plasma of healthy SD rats.

[0163] 3. Experimental Preparation

[0164] 3.1 Test product information

[0165] Table 9

[0166] 3.2 Reagent Information

[0167] Table 10

[0168] Table 11

[0169] 3.3 Animals and breeding environment

[0170] This experiment used male adult Sprague-Dawley rats, weighing 240-260 g and 6 weeks old, provided by Zhejiang Weitong Lihua Laboratory Animal Co., Ltd. The animals were housed in standard polypropylene cages at a stable temperature of 22-26°C under a 12-h light / dark cycle. The animals had free access to water and food.

[0171] 3.4 SR19 animal efficacy test preparation

[0172] 0.1 mol / L citric acid buffer: Accurately weigh 4.2 g of citric acid powder into a reagent bottle, add 150 ml of deionized water and stir until the solution is fully dissolved, adjust the pH to 4.4, make up to 200 ml, and store in a refrigerator at 4°C until used.

[0173] STZ Solution: Weigh an appropriate amount of STZ reagent and dissolve it in 0.1 mol / L citric acid buffer. Vortex until a clear solution is obtained. Note: Protect from light throughout the preparation process. Prepare immediately and inject within 10 minutes of preparation.

[0174] Solvent: Weigh 10 g of MC powder and dilute to 1 L with deionized water to prepare a 1% MC solution. Vortex and stir at room temperature overnight and store in a refrigerator at 4°C until use.

[0175] SR19 preparation: Weigh an appropriate amount of compound SR19 powder, add 1% MC solution according to the proportion, vortex and sonicate until a uniform suspension with a concentration of 1 mg / mL is formed.

[0176] LPS preparation: Weigh an appropriate amount of LPS powder and dissolve it in normal saline to prepare solutions with concentrations of 1 mg / mL, 2 mg / mL, and 5 mg / mL. Vortex and sonicate until a clear solution is formed.

[0177] 3.5 Experimental preparation of SR19 to inhibit in vitro activity of Lp-PLA2 in plasma of healthy SD rats

[0178] Preparation of experimental working solution:

[0179] 1× Assay buffer: 50 mM HEPES, 1 mM CHAPS, and 150 mM NaCl (pH adjusted to 7.4 with NaOH);

[0180] 5× Substrate solution: 2.5 mM 2-thio-PAF, 3.2 mM NEM, 0.032 mM CPM;

[0181] Termination solution: 5% TFA.

[0182] SR19 solution preparation:

[0183] SR19 was prepared in DMSO to a 10 mM stock solution. In the experiment, the 10 mM SR19 stock solution was diluted with DMSO to a 1 mM secondary stock solution. This was then serially diluted 3-fold in DMSO to obtain 10 consecutive compound concentrations. Each concentration point was then diluted 20-fold in 1× assay buffer to obtain working solutions of the compound at concentrations of 50, 16.67, 5.56, 1.85, 0.62, 0.21, 0.07, 0.02, 0.008, and 0.003 μM. The final concentrations of the compound after addition to the reaction system were: 1, 0.33, 0.11, 0.04, 0.01, 0.004, 0.001, 0.0005, 0.0002, and 0.00005 μM.

[0184] 4. Experimental methods and steps

[0185] 4.1 SR19 efficacy in animals

[0186] 4.1.1 Establishment of Type I Diabetes Rat Model

[0187] Upon arrival at the animal facility, rats were acclimated to the living environment for 2-3 days. Body weight and baseline blood glucose levels were measured after a 6-hour fast. STZ-induced diabetic rats were then injected intraperitoneally with 60 mg / kg STZ solution after a 6-hour fast. Following this, rats were given 10% glucose solution for 24 hours to prevent hypoglycemic shock and death. Fasting blood glucose was measured on day 7 after STZ injection. Rats with blood glucose levels exceeding 16.7 mmol / L were considered type 1 diabetic rats and subsequently underwent LPS-induced diabetic rats.

[0188] 4.1.2 Establishment of rat model of acute lung injury

[0189] After anesthesia, the rat's limbs were secured to a surgical board. The neck was shaved and disinfected with iodine. An approximately 1-cm incision was made in the neck, and the muscles were bluntly dissected to expose the trachea. LPS preparation or saline was slowly and evenly infused into the trachea over 30 seconds using a 1-mL syringe with a 27-gauge needle. Immediately after injection, the rat was placed upright with its head up and its tail down for 1 minute to allow for adequate fluid flow. The wound was then sutured. The rat was placed on a heating pad to maintain body temperature until it regained consciousness.

[0190] 4.1.3 Dosage Information

[0191] The dosing volume for each rat was determined based on its specific body weight, dose, and reagent concentration. The STZ dose was 60 mg / kg, and the SR19 dose was 10 mg / kg. The LPS solutions of 1 mg / mL, 2 mg / mL, and 5 mg / mL corresponded to the actual animal doses of 1 mg / kg, 2 mg / kg, and 5 mg / kg, respectively.

[0192] The specific calculation formula for the administration volume is: administration volume (mL) = body weight (kg) × (administered dose (mg / kg) / concentration of the prepared reagent (mg / mL)).

[0193] 4.1.4 Sample collection

[0194] 4.1.4.1 Plasma samples

[0195] After LPS modeling, animal mortality was recorded. Animals that survived 48 hours after LPS modeling were anesthetized and sacrificed by cardiac blood sampling. Whole blood samples were collected in K2EDTA-containing centrifuge tubes, centrifuged at 5000 rpm for 10 minutes at 4°C to separate plasma, and then stored at -80°C until analysis.

[0196] 4.1.4.2 Lung tissue samples

[0197] At the end of the experiment, the animals were anesthetized and sacrificed by cardiac exsanguination. The thoracic cavity and cervical trachea were surgically exposed. The right lung was ligated, and the left lung was intubated and fixed with 0.2 mL of 4% PFA. After ligation, the lung was placed in a tissue embedding cassette and completely immersed in 4% PFA. It was then stored at 4°C for subsequent analysis.

[0198] 4.1.5 Sample determination

[0199] 4.1.5.1 Mortality Statistics

[0200] After LPS modeling, the animal status was observed and recorded in a timely manner. There were three types of rat status: normal, the rats could eat and drink normally, their hair was neat and tidy, and their autonomous activities were normal; dying, the hair was disheveled and dull, accompanied by slow movements, slightly open eyes, fundus hemorrhage or inflammation, black nose, etc.; and death.

[0201] 4.1.5.2 Determination of Lp-PLA2 Enzyme Activity in Plasma

[0202] The assay for Lp-PLA2 activity in plasma uses 2-thio-PAF as the substrate, with absorbance readings at 405 nm and 510 nm on a multi-functional microplate reader. Lp-PLA2 activity is expressed as the amount of substrate hydrolyzed per milliliter of plasma per unit time (nmol / min / mL).

[0203] The specific method is as follows: thaw the plasma at room temperature, level it and place it in a centrifuge at 2000 rpm for 1 minute; add 20 μL of plasma to a 96-well test plate (brand: Greiner, product number: 655101), add 15 μL of 1× assay buffer to each well, and oscillate to mix well for use; negative control well (LC): according to the plate map, add 5 μL of 200 μM Darapladib solution (positive compound, dilute the compound with 1× assay buffer) to each well; for other experimental wells, according to the plate map, add 5 μL of 2% DMSO solution (diluted with 1× assay buffer) to each well; add 10 μL of 10× DTNB suspension to the test plate, oscillate to mix until the solution is clear, and incubate at room temperature for 30 minutes; after incubation, place it in a 37°C biochemical incubator for 5-10 minutes, add 50 μL The reaction was initiated by adding 2× substrate solution to the test plate. The test plate was shaken and mixed, and placed in a microplate reader with the temperature set to 37°C. Kinetic detection was performed at a wavelength of 405 / 510 nm.

[0204] 4.1.5.3 Detection of macrophage infiltration in lung tissue

[0205] Lung tissue was cut into 4 μm paraffin sections along the maximum cross-section. The sections were dewaxed, dehydrated, and permeabilized, then stained with a recombinant CD68 antibody, mounted, and scanned using a high-throughput digital slide scanner. The proportion of CD68-positive cells relative to the total number of cells in each animal's lung tissue sample was calculated using HALO digital pathology analysis software.

[0206] 4.2 In vitro inhibition of Lp-PLA2 activity by SR19 in healthy SD plasma

[0207] 1) Thaw the plasma at room temperature, balance it and place it in a centrifuge at 2000 rpm for 1 minute;

[0208] 2) Add 40 μL of plasma to each well of a 96-well assay plate (Brand: Greiner, Cat. No.: 655076) and set aside.

[0209] 3) Add 1 μL of compound working solution to each well of a 96-well assay plate according to the plate layout;

[0210] Positive control wells (HC): add 1 μL 5% DMSO (DMSO diluted with 1×Assay buffer) according to the plate layout; negative control wells (LC): add 1 μL 50 μM standard compound solution (compound diluted with 1×Assay buffer) according to the plate layout.

[0211] 4) The plasma and compound mixture was vortexed and incubated at room temperature for 30 minutes;

[0212] 5) After incubation, add 10 μL of 5× substrate solution to the 96-well assay plate to initiate the reaction;

[0213] 6) After shaking and mixing at room temperature for 2 minutes, 25 μL of 5% TFA solution was added to terminate the reaction;

[0214] 7) Place the 96-well test plate level and centrifuge at 2000 rpm for 1 minute.

[0215] 8) Place the 96-well assay plate in a multifunctional microplate reader, read the plate at a wavelength of Ex / Em = 380 / 485 nm, and collect data.

[0216] 4.3 Statistical analysis

[0217] 4.3.1 SR19 efficacy in animals

[0218] 4.3.1.1 Analysis of the effect of SR19 on mortality in ALI rats. Experimental data were analyzed and processed using Microsoft Excel 2010.

[0219] 4.3.1.2 Analysis of Lp-PLA2 activity in plasma. Experimental data were collected using Biotek Synergy H1, Software Version Gen 5, and analyzed using Microsoft Excel 2010. One-way ANOVA and Tukey's multiple comparisons test were used to analyze the differences between the groups. Statistical results are expressed as mean ± standard error. p < 0.05 indicates a significant difference in the statistical results. SR19 inhibits Lp-PLA2 activity in SD rat plasma IC 50 The values ​​were analyzed by nonlinear regression using Software Version Gen 5 software.

[0220] 4.3.1.3 Before analyzing lung tissue macrophage infiltration data, animal group information was masked, and the total cell count and CD68-positive cell count in each animal's lung tissue section were counted. The total number of CD68-positive cells in each animal's lung tissue was divided by the total number of cells, representing the percentage of CD68-positive cells in each animal's lung tissue and the presence of macrophage infiltration in each animal's lung tissue. All data were imported into Microsoft Excel 2010 for analysis and processing of the percentage of CD68-positive cells in each animal's lung tissue. Group information was then unblinded, and a two-way ANOVA was performed, with the Tukey method for multiple comparisons. Statistical results are expressed as mean ± standard error. A p < 0.05 indicated statistical significance.

[0221] 4.3.2 Inhibition of Lp-PLA2 Activity in Healthy Rat Plasma by SR19 in Vitro

[0222] The experimental data were collected using Biotek Synergy H1, Software Version Gen 5 software and analyzed using Microsoft Excel 2010.

[0223] In the experiment, experimental control conditions were defined based on the signal. The average Lp-PLA2 enzyme activity in the wells containing plasma and 0.1% DMSO was used as the positive control (defined as "High control") for no inhibition; the average Lp-PLA2 enzyme activity in the wells containing plasma and saturating concentrations of standard compounds was used as the negative control (defined as "Low control") for 100% inhibition. The inhibition rate was calculated using the following formula: Inhibition rate (%) = [1 - (experimental wells - average value)] [阴性控制] ) / (average value [阳性控制] -average value [阴性控制] )] × 100. When the number of tests per experiment was n ≥ 3, the data were expressed as the mean ± standard deviation (SD); when n < 3, the data were expressed as the mean. Nonlinear regression analysis was performed using Software Version Gen 5.

[0224] 5. Experimental results and discussion

[0225] 5.1 SR19 efficacy in animals

[0226] 5.1.1 Effect of SR19 on mortality in ALI diabetic rats

[0227] Animals were gavaged with 10 mg / kg SR19 starting one day before LPS instillation for three consecutive days. Survival was monitored for 48 hours after LPS instillation. Results showed that at a 5 mg / kg LPS dose, ALI modeling resulted in more animals dying and dying in type 1 diabetic rats. Three consecutive days of gavage with 10 mg / kg SR19 protected against mortality in rats with pre-diabetic ALI (see Table 12, Figure 5), but had a weaker protective effect against LPS-induced ALI in normal rats (see Table 12). No death occurred in diabetic rats and normal rats induced by 1 mg / kg LPS; the survival rates of ALI rats induced by 2 mg / kg LPS were: 77.78% in the LPS+Vehicle group, 80.00% in the LPS+SR19 group, 77.78% in the STZ+LPS+Vehicle group, and 100% in the STZ+LPS+SR19 group, and no rats were in a moribund state in any group; the survival rates of ALI rats induced by 5 mg / kg LPS were: 71.43% in the LPS+Vehicle group, 71.43% in the LPS+SR19 group, 60% in the STZ+LPS+Vehicle group (40% of the rats were in a moribund state and 20% of the rats were in a normal state), and 100% in the STZ+LPS+SR19 group. In Table 12, the number of animals in each group at a 1 mg / kg LPS dose (including the Naive+Vehicle and Naive+SR19 groups) was 7, the number of animals in the groups other than the LPS+SR19 group (including the Naive group) at a 2 mg / kg dose was 9, and the number of animals in the LPS+SR19 group was 10. At a 5 mg / kg dose, the number of animals in each of the Naive+Vehicle and Naive+SR19 groups was 6, the number of animals in each of the LPS+Vehicle and LPS+SR19 groups was 7, and the number of animals in each of the STZ+LPS+Vehicle and STZ+LPS+SR19 groups was 5.

[0228] Table 12 Survival rates of diabetic rats and normal rats induced by different doses of LPS

[0229] 5.1.2 Results of Lp-PLA2 enzyme activity in plasma

[0230] Plasma Lp-PLA2 activity assays showed significant increases in both 1 mg / kg LPS-induced ALI rats (p < 0.05) and STZ+LPS-induced ALI rats (p < 0.05) (Figure 6). In both the 1 mg / kg LPS-induced and STZ+LPS-induced ALI rat models, plasma Lp-PLA2 activity in the 10 mg / kg / day SR19 group decreased by 99.48 ± 0.31% (mean ± SE, n = 7) and 94.42 ± 2.27% (mean ± SE, n = 7), respectively, compared to the corresponding vehicle group. Results are shown in Table 13.

[0231] Table 13 SR19 inhibits Lp-PLA2 enzyme activity in SD rat plasma

[0232] 5.1.3 Analysis of macrophage infiltration in lung tissue

[0233] In lung tissue, CD68 antibodies can be used to mark activated and polarized macrophages. This study used the quantitative analysis method of CD68 antibody IHC staining results to explore the macrophage infiltration of lung tissue. 48 hours after LPS tracheal instillation, the results showed that the lung tissues of diabetic rats and normal rats induced by 1 mg / kg LPS showed obvious macrophage infiltration. Continuous oral administration of 10 mg / kg SR19 for 3 consecutive days had a certain inhibitory effect on the macrophage infiltration of the lung tissues of ALI rats with diabetes (see Table 14, Figure 7), and had a weak protective effect on LPS-induced normal rats (see Table 14, Figure 7). The proportions of CD68 positive cells in the lung tissues of ALI rats administered with SR19 were as follows: The percentages of CD68-positive cells in the lung tissues of ALI rats without drug administration were as follows: The LPS+Vehicle group was 8.7084±0.638% (mean±standard error, n=5), the LPS+Vehicle group was 15.9792±1.379% (mean±standard error, n=5), and the STZ+LPS+Vehicle group was 17.2754±1.149% (mean±standard error, n=5). +Vehicle, the proportion of CD68-positive cells in the lung tissues of 1 mg / kg LPS-induced ceftazidine diabetic rats and normal rats increased by 83.49±15.83% and 98.38±13.19%, respectively (mean ± standard error, n=5); compared with ALI diabetic rats not treated with SR19, the proportion of CD68-positive cells in the lung tissues of ALI diabetic rats treated with SR19 decreased by 24.45±7.37% (mean ± standard error, n=5), p=0.06.

[0234] Table 14. Percentage of CD68 positive cells in rat lung tissue

[0235] 5.2 Inhibition of Lp-PLA2 Activity in Healthy SD Rat Plasma by SR19 in Vitro

[0236] As shown in the table below, the IC value of SR19 on Lp-PLA2 enzyme activity in the plasma of healthy SD rats is 50 The average value was 12.25 nM (n=2).

[0237] Table 15. Summary of the inhibition data of SR19 on Lp-PLA2 activity in plasma of healthy SD rats

[0238] In summary, this experimental example compared the harmful effects of lipopolysaccharide (LPS) induced acute lung injury (ALI) in normal rats and type 1 diabetic rats after intratracheal instillation, and on this basis evaluated the protective effect of SR19 on these two types of ALI rats. The results showed that ALI modeling caused a higher mortality rate in type 1 diabetic rats, and SR19 was able to reduce the mortality caused by ALI modeling in type 1 diabetic rats and improve the macrophage infiltration of lung tissue. Both normal rats and type 1 diabetic rats showed a significant increase in plasma Lp-PLA2 enzyme activity after ALI modeling. After administration of 10mg / kg / day SR19, the Lp-PLA2 activity in the plasma of the two rats decreased by 99.48% and 94.42%, respectively, compared with the vehicle group (Vehicle).

[0239] Experimental Example 3: Study on the efficacy of SR15 in a mouse model of acute pneumonia induced by nasal instillation of Pseudomonas aeruginosa

[0240] 1. Introduction

[0241] Pneumonia is a common acute respiratory infection, broadly categorized as community-acquired pneumonia and hospital-acquired pneumonia. Disease progression depends largely on the host's immune response, and excessive production of proinflammatory cytokines is a major factor in accelerating the progression of pneumonia to severe illness or acute respiratory distress syndrome (ARDS). Therefore, timely blocking the production of proinflammatory cytokines may help prevent pneumonia from progressing to severe illness or ARDS.

[0242] Lipoprotein-associated phospholipase A2 (Lp-PLA2) is expressed in pneumonia (see Li Y, Jiang Y, Zhang Y, et al. Abnormal upregulation of cardiovascular disease biomarker PLA2G7 induced by proinflammatory macrophages in COVID-19 patients. Sci Rep. 2021; 11(1): 6811.) and lung diseases (COPD (see Mingming Deng, et al. Identification of Inflammation-Related Biomarker Lp-PLA2 for Patients With COPD by Comprehensive Analysis. Front Immunol. 2021 May 21; 12: 670971.) and IPF (Junyi Wang, et al. Integrated analysis of single-cell and bulk RNA sequencing reveals pro-fibrotic PLA2G7 high macrophages in pulmonary fibrosis. Pharmacol Res. 2022 Aug;182:106286.)) It is highly expressed in nasal swabs and lung macrophages of patients, its plasma level is significantly increased, and is positively correlated with the severity of the disease. Lp-PLA2 levels are also abnormally elevated in COVID-19 patients, and Lp-PLA2 levels return to normal when fully recovered. Lysophosphatidylcholine (LysoPC) is one of the main bioactive products of Lp-PLA2, with multiple pro-inflammatory activities. As a monocyte chemotactic factor, it induces oxidative stress and endothelial dysfunction and upregulates cytokine (IL-1β, IL-6, TNF-α) expression (see Fubao Huang, Kai Wang, et al. Lipoprotein-associated phospholipase A2: The story continues. Med Res Rev. 2020 Jan;40(1):79-134.).

[0243] Pseudomonas aeruginosa (PA) is a Gram-negative bacterium that secretes multiple virulence factors, including endotoxins (lipopolysaccharides), exotoxins, and elastase, and is widely present in nature. The PA-induced acute pneumonia mouse model effectively simulates the physiological processes of inflammation and lung injury that occur after acute pneumonia. It is clinically relevant and relatively safe and easy to operate (see Varsha Suresh Kumar et al. Pseudomonas aeruginosa Induced Lung Injury Model. J Vis Exp. 2014;(92):52044). Recent studies suggest that the prevalence of Pseudomonas aeruginosa community-acquired pneumonia is 67% in patients with chronic lung disease and established P. aeruginosa infection (Restrepo MI, Babu BL, Reyes LF, Chalmers JD, Soni NJ, Sibila O, et al. Glimp. Burden and risk factors for Pseudomonas aeruginosa community-acquired pneumonia: a multinational point prevalence study of hospitalized patients. Eur Respir J. 2018;52:1701190.). This study aimed to evaluate the efficacy of SR15A in a mouse model of acute pneumonia induced by intranasal instillation of P. aeruginosa.

[0244] 2. Experimental Design

[0245] The experimental design is shown in Figure 8. After the experimental mice (C57BL / 6 mice, 8-10 weeks old, male) arrived at the facility and adapted to the environment for 1 week, they were divided into 5 groups: Control, PA mice + vehicle, PA mice + 10 mg / kg SR15, PA mice + 50 mg / kg CAZ (Ceftazidime), and PA mice + 10 mg / kg SR15 / 50 mg / kg CAZ.

[0246] 10 mg / kg SR15 was administered orally twice a day (12 h + 12 h) for three consecutive days. On the second day, the bacterial solution was instilled into the nasal cavity 1 hour after the first administration. PA (1×10 7Plasma, bronchoalveolar lavage fluid (BALF), and lung tissue were collected 48 hours after PA induction (using CFU, ATCC 27853). Plasma Lp-PLA2 activity, cytokine levels, and inflammatory cell levels in BALF were measured, and lung tissue pathology was analyzed. Because Lp-PLA2 inhibitors lack antibacterial or bactericidal properties, their efficacy was evaluated in combination with an antibiotic (CAZ). The antibiotic was administered twice daily (12 hours + 12 hours) 12 hours after PA induction. Plasma Lp-PLA2 activity was measured enzymatically using thio-PAF as a substrate. BALF cytokine levels were measured using enzyme-linked immunosorbent assay (ELISA). BALF inflammatory cell counts were determined using a hematology analyzer. Lung tissue was analyzed by single-blind hematoxylin and eosin (HE) staining.

[0247] 3. Experimental results

[0248] 3.1. SR15 significantly inhibited the increase in plasma Lp-PLA2 activity in mice with acute pneumonia induced by PA

[0249] In a mouse model of acute pneumonia induced by PA, plasma Lp-PLA2 activity was significantly elevated. CAZ had no effect on plasma Lp-PLA2 activity, while SR15 or SR15 / CAZ inhibited plasma Lp-PLA2 activity by greater than 90%. See Figure 9 and Table 16 for details.

[0250] Table 16 Inhibitory effect of SR15 on Lp-PLA2 activity in plasma of mice with PA-induced acute pneumonia

[0251] SR15 inhibits chemokine secretion and inflammatory cell infiltration in BALF of mice with PA-induced acute pneumonia

[0252] The results are shown in Figure 10. The level of monocyte chemoattractant protein (MCP-1) in the BALF of mice with acute pneumonia induced by PA was significantly increased. The inhibition rates of 10mg / kg SR15, 50mg / kg CAZ, and SR15 combined with CAZ on MCP-1 in BALF were 27.2%, 36.8%, and 50.0%, respectively. At the same time, the levels of neutrophils and monocytes in BALF were significantly increased. Neither SR15 nor CAZ showed an inhibitory effect on neutrophils in BALF when used alone. The inhibition rate after SR15 and CAZ combined was 28.1%; the inhibition rate of 50mg / kg CAZ on monocytes in BALF was 14.4%. The inhibition rate after SR15 combined with CAZ was 37.2%, which was 22.8% higher than the inhibition rate of CAZ alone.

[0253] 3.3. SR15 improves lung tissue pathological damage in mice with PA-induced acute pneumonia

[0254] Mice infected with PA showed significant pathological damage in their lung tissue, and the severity of the damage was scored using multiple dimensions. Each dimension of the HE score is scored as follows: 0 (absent); 1 (minor); 2 (major). The results, as shown in Figure 11, showed that for interstitial and alveolar hemorrhage, SR15 alone improved by 40%, CAZ alone improved by 66.6%, and the combined treatment improved by 66.6%. For perivascular edema, SR15 and CAZ alone improved by 16.7%, while the combined treatment increased this to 44.4%. For interstitial inflammation, SR15 alone improved by 33.3%, while CAZ alone showed no improvement. Compared to CAZ alone, the combined treatment showed a 37.5% improvement. For perivascular inflammation, SR15 alone improved by 20%, while CAZ alone showed no improvement. Compared to CAZ alone, the combined treatment showed a 50% improvement. The results suggest that SR1 has a certain improvement effect on lung tissue pathological damage and can be used in combination with antibiotics to further enhance the improvement of lung tissue pathological damage.

[0255] In summary, in a mouse model of acute pneumonia induced by a microorganism (Pseudomonas aeruginosa), plasma Lp-PLA2 activity was significantly elevated. This study demonstrates for the first time in a bacterial microorganism-induced acute pneumonia model that an Lp-PLA2 inhibitor (SR1) can effectively inhibit chemokine secretion and inflammatory cell infiltration in the BALF of mice with pneumonia. While the drug alone has a modest effect on lung tissue pathology, combined with antibiotics, it further enhances this effect.

[0256] Experimental Example 4: Study on the efficacy of SR19 on the acute lung injury model of COPD rats induced by smoke combined with LPS

[0257] 1. Introduction

[0258] Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory disease characterized by persistent and progressive airflow limitation.

[0259] Lipoprotein-associated phospholipase A2 (Lp-PLA2) is highly expressed in lung diseases (COPD and IPF (see Junyi Wang, et al. Integrated analysis of single-cell and bulk RNA sequencing reveals pro-fibrotic PLA2G7 high macrophages in pulmonary fibrosis. Pharmacol Res. 2022 Aug; 182: 106286.)) and pulmonary macrophages. Its plasma level is significantly increased and positively correlated with the GOLD grade of COPD patients and negatively correlated with the lung function FEV1 / FVC (%) of COPD patients. Lysophosphatidylcholine (LysoPC) is one of the main bioactive products of Lp-PLA2 and has multiple pro-inflammatory activities. As a monocyte chemotactic factor, it induces oxidative stress and endothelial dysfunction and upregulates cytokine (IL-1β, IL-6, TNF-α) expression (see Fubao Huang, Kai Wang, et al. Lipoprotein-associated phospholipase A2: The story continues. Med Res Rev. 2020 Jan; 40(1): 79-134.).

[0260] The COPD acute lung injury rat model induced by cigarette smoke combined with lipopolysaccharide (LPS) exhibits pathological changes such as bronchial mucosal epithelial damage at all levels, increased airway secretions, airway airflow obstruction, and inflammatory cell infiltration in the airways and lung parenchyma (see Yi-Chu Nie et al. Characteristic comparison of three rat models induced by cigarette smoke or combined with LPS: to establish a suitable model for study of airway mucus hypersecretion in chronic obstructive pulmonary disease. Comparative Study Pulm Pharmacol Ther. 2012 Oct; 25(5): 349-56.), which can model the clinical phenomena of acute exacerbation of COPD patients. This study aimed to evaluate the efficacy of SR19 on the COPD acute lung injury rat model induced by cigarette smoke combined with LPS.

[0261] 2. Experimental Design

[0262] 2.1 Experimental animals and groups

[0263] After the experimental rats (SD rats, 8-10 weeks old, male) arrived at the facility and adapted to the environment for one week, they were divided into two groups: a model control group (Vehicle) and a model administration group (SR19).

[0264] 2.2 Experimental process of smoke fumigation combined with LPS airway atomization

[0265] The experimental process is shown in Figure 12. On Day 0, after the Penh lung function test, ~500 μl of whole blood was collected venously and placed in an EP tube containing the anticoagulant EDTA·K2. Subsequently, the blood was centrifuged at 3500 rpm, 4°C, for 10 min to prepare 100 μl of plasma in two tubes. The prepared plasma was frozen at -80°C for future use.

[0266] From Day 1 to Day 38, rats were exposed to cigarette smoke (Huangguoshu cigarettes, manufactured by Guizhou China Tobacco Industry Co., Ltd.; tar content: 10 mg; smoke nicotine content: 0.9 mg; smoke carbon monoxide content: 12 mg). Exposures were conducted twice daily, one hour in the morning and one in the afternoon, with 20 cigarettes each time. Blood was collected on Day 7 and Day 14 for measurement of Lp-PLA2 and oxLDL levels. LPS (2.5 mg / kg and 2 mg / kg, respectively) was administered via aerosolized airway before smoking on Day 10 and Day 25. Doses are shown in Figure 12.

[0267] 2.3 Pulmonary function test

[0268] Rats were placed in the plethysmography chamber of the DSI Buxco WBP system on Day 0 (before smoking) before smoke + LPS modeling, Day 23 (before drug administration) after modeling, Day 30 (7 days after drug administration) and Day 38 (14 days after drug administration) after modeling to detect the enhanced expiratory pause (PenH) of the animals in the awake state.

[0269] 2.4 Animal Dosing Regimen and Endpoint Sample Collection

[0270] On Day 24, rats were divided into groups based on body weight, Penh, and other indicators. Each group was treated with the corresponding vehicle or compound for 14 consecutive days from Day 24 to Day 38. At the end of the experiment, BALF samples were collected and analyzed for leukocyte differential analysis and lysophosphatidylcholine (LysoPC) levels.

[0271] 3. Experimental results

[0272] 3.1 Results of plasma Lp-PLA2 and oxLDL levels

[0273] The levels of Lp-PLA2 and oxLDL in the plasma of rats with acute lung injury induced by smoke combined with LPS were significantly increased (see Figure 13 for details).

[0274] 3.2. Leukocyte count and LysoPC analysis results in BALF

[0275] The results of white blood cell counts showed that compared with the model control group (vehicle), the 10 mg / kg SR19 administration group had a 58.6% decrease in monocyte infiltration in the BALF.

[0276] The results of LysoPC lipid metabolism showed that the concentrations of LysoPC (16:1), LysoPC (18:1), LysoPC (18:2), LysoPC (20:4), LysoPC (22:6) and LysoPC (24:0) in the model control group (Vehicle) were significantly higher than those in the control group (Vehicle). The control group (Naive control) was significantly increased. The inhibition rates of LysoPC in BALF in the 10 mg / kg SR19-treated group were 40.3%, 20.5%, 40.5%, 59.8%, 25.2% and 79.4%, respectively. In the acute lung injury model induced by smoke combined with LPS, the type of LysoPC inhibited by SR19 was consistent with the type of LysoPC upregulated in the lung epithelial cells of COPD patients (see Abilash Ravi et al. Metabolic differences between bronchial epithelium from healthy individuals and patients with asthma and the effect of bronchial thermoplasty. Clinical Trial J Allergy Clin Immunol. 2021 Nov; 148(5): 1236-1248.). The results are shown in Figure 15.

[0277] 3.3. SR19 significantly improved lung function in rats with acute lung injury induced by smoke combined with LPS

[0278] Pulmonary function in experimental rats was measured using the DSI Buxco WBP system's plethysmography system, with results expressed as PenH. Results showed that before SR19 administration, baseline PenH levels in the vehicle and SR19-treated groups were essentially identical (Day 23). Seven days after SR19 administration, PenH levels in the vehicle control group continued to rise. However, the SR19-treated group significantly suppressed the increase in PenH levels (P<0.01), demonstrating a continuous downward trend compared to pre-drug administration. The results are shown in Figure 16.

[0279] In summary, smoke combined with LPS-induced acute lung injury in COPD rats significantly elevated both plasma levels of Lp-PLA2 and its substrate, oxLDL. This study demonstrates for the first time in this COPD rat model of acute lung injury that SR19 significantly inhibits Lp-PLA2 hydrolysis of oxLDL into the proinflammatory product LysoPC, inhibiting pulmonary monocyte infiltration and significantly improving lung function. This demonstrates for the first time the therapeutic potential of Lp-PLA2 inhibitors in patients with acute exacerbations of COPD.

[0280] Experimental Example 5: Study on the efficacy of SR13 on the acute lung injury model induced by LPS tracheal aerosol in diabetic rats

[0281] 1. Introduction

[0282] Elderly people with chronic underlying diseases are the most affected by pneumonia. Chronic underlying diseases increase susceptibility to pneumonia and delay its resolution; pneumonia can exacerbate chronic underlying diseases, increasing the likelihood of severe and critical pneumonia in patients with chronic underlying diseases [see Lee J Quinton et al. Integrative Physiology of Pneumonia. Physiol Rev. 2018 Jul 1; 98(3):1417-1464.].

[0283] Lipoprotein-associated phospholipase A2 (Lp-PLA2) is significantly elevated in the plasma of patients with chronic underlying diseases (diabetes, cardiovascular disease, chronic obstructive pulmonary disease (COPD), etc.); it is highly expressed in nasal swabs and lung macrophages of patients with pneumonia and COVID-19, its plasma level is significantly elevated, and is positively correlated with the severity of the disease [see Anolin Aslan, Cynthia Aslan, et al. Acute respiratory distress syndrome in COVID-19: possible mechanisms and therapeutic management. Pneumonia (Nathan). 2021 Dec 6; 13(1): 14]. Lysophosphatidylcholine (LysoPC) is one of the main bioactive products of Lp-PLA2, with multiple pro-inflammatory activities, inducing oxidative stress and endothelial dysfunction [see Fubao Huang, Kai Wang, et al. Lipoprotein-associated phospholipase A2: The story continues. Med Res Rev. 2020 Jan; 40(1): 79-134.].

[0284] Studies have shown that delayed lung tissue repair in a rodent model of LPS-induced acute lung injury under diabetic conditions can mimic clinical phenomena such as poor prognosis and prolonged recovery time after pneumonia in patients with chronic underlying diseases [see Yoshiro Yamashita et al. Impairment of tissue repair in pneumonia due to β-cell deficiency: role of endoplasmic reticulum stress in alveolar macrophages. BMC Res Notes. 2019 Mar 22]. SR13 is an oral, selective Lp-PLA2 inhibitor. This study aimed to evaluate the efficacy of SR1375A in a LPS nebulized tracheal aerosol-induced acute lung injury model in diabetic rats.

[0285] 2. Experimental Design

[0286] 2.1 Experimental design for evaluating the time-dependent efficacy of SR13

[0287] After acclimation to the facility for 2-3 days, rats (SD adult males, 6-8 weeks old) were injected intraperitoneally with STZ solution (60 mg / kg) to induce a diabetic model. Based on the diabetic rat model, an acute lung injury model (STZ / LPS ALI) was induced by intratracheal aerosol injection of LPS.

[0288] Animals were collected for endpoint analysis on day 3 (LPS-Day 3) and day 5 (LPS-Day 5) after LPS induction, as shown in Table 17. Animals in both groups (STZ / LPS-Day 3 and STZ / LPS-Day 5) were divided into three experimental groups: STZ / LPS + Vehicle and STZ / LPS + SR13. The SR13 dose was 10 mg / kg. SR13 and vehicle were administered by single oral gavage daily starting one day before LPS induction for 3 and 5 consecutive days (see Figure 17). At the end of the experiment, BALF and lung tissue were collected for subsequent analysis.

[0289] Table 17 Animal groups and dosing regimens for the in vivo efficacy experiment of SR13

[0290] 2.2 Experimental design for evaluating the dose-dependent efficacy of R13

[0291] The STZ / LPS ALI model refers to Section 2.1 of Experimental Example 5. The experimental animals were divided into 6 experimental groups: STZ, STZ / LPS + Vehicle, STZ / LPS + 1 mg / kg SR13, STZ / LPS + 3 mg / kg SR13, and STZ / LPS + 10 mg / kg SR13 were administered daily by single gavage starting one day before LPS induction for five consecutive days (see Table 18). At the experimental endpoint, plasma and BALF were collected on day 5 after the single LPS induction for subsequent analysis (see Figure 18).

[0292] Table 18 Animal groups and dosing regimens for the in vivo efficacy experiment of SR13

[0293] Plasma samples were tested for Lp-PLA2 enzyme activity and SR1375A concentration. Plasma samples were shipped on dry ice to Shanghai Jingtai Biotechnology Co., Ltd. for plasma drug concentration testing. BALF samples were processed, placed in a wet ice box, and sent the same day to Shanghai Bikekoyi Biotechnology Co., Ltd. for white blood cell differential analysis. Lung tissue was placed in a 50mL centrifuge tube filled with 4% paraformaldehyde (PFA), placed in a foam box with ice packs, and mailed to Wuhan Saiweier Biotechnology Co., Ltd. for pathological analysis.

[0294] 3. Experimental Preparation

[0295] 3.1 Test product information

[0296] Table 19. Test product information

[0297] 3.2 Reagent Information

[0298] Table 20, Reagent Information

[0299] 3.3 Main instruments and equipment

[0300] Table 21. Main instruments and equipment

[0301] 3.4 Animals and Breeding Environment

[0302] This experiment used male adult Sprague-Dawley rats weighing 280–330 g, provided by Zhejiang Weitong Lihua Laboratory Animal Co., Ltd. The animals were housed in standard polypropylene cages at a stable temperature of 22–24°C with a 12-h light / dark cycle. The animals had free access to water and food, and their cages were disinfected and changed regularly.

[0303] 3.5 Experimental Preparations

[0304] 0.1 mol / L citric acid buffer: Accurately weigh 4.2 g of citric acid powder into a reagent bottle, add 150 ml of deionized water and stir until the solution is fully dissolved, adjust the pH to 4.4, make up to 200 ml, and store in a refrigerator at 4°C until used.

[0305] STZ Solution: Weigh an appropriate amount of STZ reagent and dissolve it in 0.1 mol / L citrate buffer. Vortex until a clear solution is obtained. Note: Protect from light and keep on ice throughout the preparation process. Prepare immediately and inject within 10 minutes of preparation.

[0306] Solvent: Weigh 10 g of MC powder and dilute to 1 L with deionized water to prepare a 1% MC solution. Vortex and stir at room temperature overnight and store in a refrigerator at 4°C until use.

[0307] LPS preparation: Weigh an appropriate amount of LPS powder and dissolve it in 1× PBS solution to prepare a solution with a concentration of 7.5 mg / mL. Vortex and sonicate until a clear solution is formed.

[0308] SR13 preparation: Weigh an appropriate amount of compound SR13 powder and add 1% MC solution according to the proportion. Vortex and sonicate until a uniform suspension is formed.

[0309] 4. Experimental methods and steps

[0310] 4.1. Establishment of the acute lung injury model in diabetic rats

[0311] Upon arrival at the animal facility, rats were acclimated to the living environment for 2-3 days. Body weight and baseline blood glucose levels were measured after a 6-hour fast. Subsequently, STZ induction was performed. After a 6-hour fast, rats were intraperitoneally injected with 60 mg / kg STZ solution. 10% sucrose was then administered for 24 hours to prevent hypoglycemic shock and death. Seven days after STZ injection, fasting blood glucose levels were measured, and rats with blood glucose levels greater than 11 mmol / L were defined as diabetic. Rats with blood glucose levels below 11 mmol / L received a second STZ injection. One week after the injection, fasting blood glucose levels were retested, and diabetic rats underwent subsequent LPS induction. After isoflurane anesthesia, the rats were secured on an intubation platform. A nebulizer needle was inserted through the mouth into the trachea using a laryngoscope. The syringe plunger was rapidly pushed to deliver LPS into the lungs as an aerosol. Following the injection, the rats were carefully observed to prevent suffocation and returned to the housing room upon recovery.

[0312] 4.2 Dosage Information

[0313] The LPS administration volume was 200 μL. The SR13 administration volume for each rat was based on its specific body weight. The standard SR13 administration volume was 10 mL / kg. The actual animal doses corresponding to 0.1 mg / mL, 0.3 mg / mL, and 1 mg / mL SR13 solutions were 1 mg / kg, 3 mg / kg, and 10 mg / kg, respectively. The LPS administration dose was 5 mg / kg. The administration volume for each rat was based on its body weight and the concentrations of the prepared SR13 suspension and LPS solution. The administration volume calculation formula is: Administration volume (mL) = body weight (kg) × (Administered dose (mg / kg) / (SR13 or LPS) concentration (mg / mL)).

[0314] 4.3 Sample Collection

[0315] 4.3.1 Blood sample collection

[0316] At the end of the experiment, whole blood samples were collected in centrifuge tubes containing EDTA-K2, centrifuged at 5000 rpm for 10 minutes (4°C) to separate the plasma, and then stored in a -80°C refrigerator until analysis.

[0317] 4.3.2 BALF supernatant samples and BALF cell collection

[0318] At the end of the experiment, the animals were anesthetized and sacrificed by blood sampling from the abdominal aorta. The thoracic cavity and cervical trachea were then surgically exposed. A slipknot was tied in the left bronchus with a suture. 2.5 mL of pre-chilled saline was injected into the right lobe with gentle pressure to lavage the lung. Two gentle aspirations were repeated, and the lavages were combined and placed on ice. The alveolar lavage fluid was centrifuged at 1500 rpm for 10 minutes at 4°C. After centrifugation, the BALF supernatant was carefully aspirated, transferred to new centrifuge tubes, and aliquoted into 1.0 mL tubes. The tubes were stored at -80°C. Samples with a BALF recovery rate greater than 80% were used for subsequent experiments. After the supernatant was transferred, the cell pellet was immediately resuspended in 500 μL of saline. The suspension was transferred to a 1.5 mL centrifuge tube and stored on ice. The samples were shipped to Shanghai Bikeway Biotechnology Co., Ltd. the same day for leukocyte differential analysis.

[0319] 4.3.3 Lung tissue collection

[0320] After obtaining the alveolar lavage fluid, the left lung slipknot was opened, and 0.5 mL of 4% PFA tissue fixative was injected into the left lung. The left alveoli were then ligated and removed and fixed in PFA solution. The left lung was placed in a 50 mL centrifuge tube filled with PFA and mailed to Wuhan Sevier Biotechnology Co., Ltd. on the same day for HE staining and pathological analysis.

[0321] 4.4 Sample determination

[0322] 4.4.1. Plasma SR13 concentration determination: Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to determine the concentration of SR13 in SD rat plasma. The test was performed by Shanghai Jingtai Biotechnology Co., Ltd.

[0323] 4.4.2. Plasma Lp-PLA2 Activity Assay: Kinetic analysis was performed using 2-thio-PAF as the substrate, with absorbance readings at 405 nm and 510 nm on a multifunctional microplate reader. Lp-PLA2 activity was expressed as the amount of substrate hydrolyzed per milliliter of plasma per unit time (nmol / min / mL).

[0324] 4.4.3. BALF albumin test: BALF albumin levels were measured using enzyme-linked immunosorbent assay (ELISA) using a multifunctional microplate reader at wavelengths of 450 nm and 510 nm.

[0325] 4.4.4. White blood cell differential count in BALF: The test was performed automatically using the XT-2000i fully automatic hematology analyzer, and the test was completed by Shanghai Bikeway Biotechnology Co., Ltd.

[0326] 4.4.5. Lung tissue pathology analysis: Wuhan Saiweier Biotechnology Co., Ltd. will perform the pathology analysis. This analysis is double-blind. The lung tissue pathology scoring standard is based on a four-level grading system, as shown in Table 22:

[0327] Table 22 Four-level grading system for lung tissue HE staining pathological scoring

[0328] Peter Mann et al. International Standards of Terminology and Diagnostic Criteria for Pathological Changes in Rats and Mice (INHAND). Translated by Yang Lifeng, Zhou Xiangmei, and Zhao Deming. Beijing: China Agriculture Press, 2019.

[0329] 4.5 Statistical Analysis

[0330] 4.5.1. PK results The experimental data were described using mean (Mean) and standard deviation (SD) using Microsoft Excel 2010.

[0331] 4.5.2 Analysis of Lp-PLA2 activity in plasma. Experimental data were collected using Biotek Synergy H1, Software Version Gen 5 software and analyzed using GraphPad Prism 9 and Microsoft Excel 2010. Statistical analysis was performed using the "Ordinary one-way ANOVA, Dunnett's multiple comparisons test" in GraphPad Prism 9 to analyze the differences between the groups. Statistical results are expressed as mean ± standard error, and p < 0.05 indicates a significant difference. SR13 inhibits Lp-PLA2 activity in plasma of STZ / LPS ALI rats IC 50 The values ​​were analyzed by nonlinear regression using GraphPad Prism 9 (log (inhibitor) vs. response–Variable slope).

[0332] 4.5.3. BALF albumin levels and differential leukocyte counts were analyzed using GraphPad Prism 9 and Microsoft Excel 2010 software. Differences between groups were analyzed using the "Ordinary one-way ANOVA, Dunnett's multiple comparisons test" in GraphPad Prism 9. Statistical results are expressed as mean ± standard error. A p < 0.05 indicated statistical significance.

[0333] 4.5.4 Lung Tissue Pathology Analysis: Scoring data for pathological injury indicators for each lung tissue were analyzed using Microsoft Excel and GraphPad Prism 5. Statistical analysis was performed using a two-way ANOVA with multiple comparisons among the control, model, and treatment groups. Data are presented as mean and standard error. P < 0.05 indicated statistical significance.

[0334] 5. Experimental results and discussion

[0335] 5.1. PK testing in plasma samples

[0336] After continuous administration of the test drug SR13 to the diabetic acute lung injury model, blood samples were collected and analyzed using the LC-MS / MS method to determine the plasma concentration of SR13 in the samples.

[0337] The results showed that after continuous gavage of 1 mg / kg, 3 mg / kg and 10 mg / kg SR13 for 5 days, the average concentrations 20 hours after the last administration were 4.49, 22.15 and 122.26 ng / mL, respectively, as shown in Table 23.

[0338] Table 23 SR13 concentration in SD rat blood

[0339] 5.2 Results of plasma Lp-PLA2 activity determination

[0340] Plasma Lp-PLA2 activity testing showed significant increases in plasma Lp-PLA2 activity in STZ / LPS ALI rats (p<0.001). Compared to the vehicle group (STZ / LPS + Vehicle), plasma Lp-PLA2 activity decreased by 34.40%, 39.47%, and 68.88% in the 1, 3, and 10 mg / kg / day SR13 groups, respectively. SR13 inhibited plasma Lp-PLA2 activity in a dose-dependent manner, as shown in Table 24 and Figure 19.

[0341] Table 24 SR13 inhibits Lp-PLA2 activity in plasma of STZ / LPS ALI rats

[0342] 5.3. SR13 inhibits inflammatory cell infiltration and albumin leakage in BALF of STZ / LPS ALI rats

[0343] In the SR13 time-dependent efficacy evaluation experiment, the total leukocyte level in BALF was compared with that in The 10 mg / kg SR13 group showed a significant increase in BALF leukocyte levels compared to the STZ / LPS + Vehicle group (LPS-Day 3). Compared to the STZ / LPS + Vehicle group (LPS-Day 3), the 10 mg / kg SR13 group showed a 38.27% decrease in overall BALF leukocyte levels. Five days after LPS induction, the total BALF leukocyte level decreased significantly compared to three days after induction, and lung inflammatory cell infiltration decreased. Compared to the STZ / LPS + Vehicle group (LPS-Day 5), the 10 mg / kg SR13 group showed a 24.71% decrease in lung inflammatory cell infiltration compared to the STZ / LPS + Vehicle group (LPS-Day 5), further reducing lung inflammatory cell infiltration (see Table 25 and Figure 20).

[0344] Compared with the white blood cell level in BALF of vehicle group The formula for calculating the group increase ratio is: The calculation formula of the inhibition rate of lung leukocyte infiltration in the drug-treated group is: inhibition rate (%) = 100-leukocyte concentration of each sample [SR13] / average leukocyte concentration of samples [Vehicle] ×100.

[0345] Table 25 SR13 inhibits lung leukocyte infiltration

[0346] In a dose-dependent efficacy study evaluating SR13, compared with the STZ / LPS+Vehicle group, the 1 mg / kg, 3 mg / kg, and 10 mg / kg SR13 groups showed a decrease in BALF monocyte counts by 17.14%, 26.19%, and 51.65%, respectively. The 3 mg / kg and 10 mg / kg SR13 groups showed a decrease in BALF lymphocyte counts by 5.84% and 23.83%, respectively. SR13 inhibited the infiltration of monocytes, macrophages, and lymphocytes in the BALF in a dose-dependent manner. Furthermore, the 1 mg / kg and 10 mg / kg SR13 groups showed a decrease in BALF neutrophil counts by 30.52% and 19.83%, respectively (see Table 26 and Figure 21).

[0347] Table 26 Inhibitory effect of SR13 on pulmonary monocytes / lymphocytes / neutrophils

[0348] In the SR13 dose-dependent efficacy evaluation experiment, the total albumin level in BALF was also measured to evaluate the inhibitory effect of SR13 on alveolar-capillary barrier destruction under modeling conditions. In the STZ / LPS ALI rat experiment, the albumin level in BALF of the Vehicle group was significantly higher than that of the control group 5 days after LPS stimulation. Compared with the vehicle group, BALF albumin levels in the 1 mg / kg, 3 mg / kg, and 10 mg / kg SR13 groups decreased by 31.38%, 27.86%, and 37.54%, respectively. The results showed that 10 mg / kg SR13 significantly inhibited albumin leakage from the BALF and suppressed the breakdown of the alveolar-capillary barrier (see Table 27 and Figure 22).

[0349] Table 27 SR13 inhibits albumin leakage in BALF

[0350] 5.4. SR13 improves lung tissue pathological damage in STZ / LPS ALI rats

[0351] Pathological examination revealed mild alveolar wall thickening in the control group three days after LPS induction, accompanied by a small infiltration of inflammatory cells, primarily neutrophils, and mild pulmonary edema in some samples. Compared with the control group, the model group showed more severe and extensive alveolar wall thickening, with unclear alveolar structure. Alveolar epithelial cell damage was evident, with increased infiltration of inflammatory cells, primarily monocytes, neutrophils, and macrophages. Pulmonary edema worsened, with an increased number of samples presenting. Mild vascular damage was also observed in some samples. Compared with the model group, the treatment group showed mild improvement in lesions, but no statistically significant differences were observed in individual lesion parameters (see Table 25).

[0352] Five days after LPS induction, some samples in the control group showed mild mononuclear cell infiltration and alveolar wall thickening. The model group had significant lung inflammation, but compared with the model group three days after LPS induction, the alveolar wall thickening was less severe and less extensive; the degree of inflammatory cell infiltration was reduced, vascular damage disappeared, alveolar epithelial cell damage was alleviated, pulmonary edema was slightly aggravated, and the overall injury score decreased, indicating that the model had some natural improvement. Five days after LPS induction, the degree of alveolar wall thickening and alveolar epithelial damage in the drug-treated group was less severe and less extensive than in the model group; the degree of inflammatory cell infiltration was reduced, and all pathological changes in the drug-treated group were alleviated compared with the model group, but no statistical differences were found in individual pathological indicators (see Table 28).

[0353] Analysis of the overall lung tissue pathology score showed that 5 days after LPS induction, the drug group had a 43.24% improvement rate compared to the model group, which was 20.94% higher than the natural improvement rate. Statistical analysis showed that the drug significantly improved lung tissue pathology lesions 5 days after LPS induction (p < 0.01) using a 2-way ANOVA multiple comparisons analysis (see Table 29 and Figure 23).

[0354] Table 28 Analysis results of various lung tissue injury indicators

[0355] Note: Administration group: 10 mg / kg SR13.

[0356] Table 29 Analysis results of overall score of lung tissue injury

[0357] Note: The formula for calculating the improvement rate of lung tissue pathological damage is: Improvement rate (%) = (score 模型组 -score 给药组 ) / score 模型组 *100, natural improvement rate (%) = (scoring model group - LPS - Day 3 - scoring model group - LPS - Day 5) / scoring model group - LPS - Day 3 * 100, drug administration group improvement rate (%) = (scoring model group - LPS - Day 3 - scoring drug administration group - LPS - Day 5) / scoring model group - LPS - Day 3 * 100. Note: The drug administration group received 10 mg / kg SR13.

[0358] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Use of the compound of formula I, its cis-trans isomers, its enantiomers, its diastereomers, its racemates, its solvates, its hydrates, its pharmaceutically acceptable salts or its prodrugs in the preparation of drugs for preventing, alleviating, assisting in the treatment or treating lung injury or in preventing, alleviating, assisting in the treatment or treating lung injury; R is H, fluoro or cyano; X is N or CR1, and R1 is H.

2. The use according to claim 1, characterized in that The compound represented by formula I is:

3. Use of the compound of formula I as described in any one of claims 1 to 2, its cis-trans isomers, its enantiomers, its diastereomers, its racemates, its solvates, its hydrates or its pharmaceutically acceptable salts or their prodrugs in combination with ceftazidime in the preparation of a medicament for preventing, alleviating, assisting in the treatment or treating lung injury or in the prevention, alleviating, assisting in the treatment or treating lung injury.

4. The use according to any one of claims 1 to 3, characterized in that: The lung injury includes: acute lung injury or its combined diseases; or acute respiratory distress syndrome or its combined diseases; or chronic obstructive pulmonary disease or its combined diseases; Optionally, the combined disease is diabetes; Optionally, the diabetes is type 1 diabetes.

5. The use according to any one of claims 1 to 4, characterized in that: The lung injury is caused by infection, pro-inflammatory cytokine storm and / or inflammatory cell infiltration.

6. The use according to claim 5, characterized in that The infections include microbial infections; Optionally, the microbial infection includes viral infection or bacterial infection; Optionally, the viral infection is a coronavirus infection; Optionally, the coronavirus infection is SARS-CoV-2 virus infection.

7. The use according to claim 5 or 6, characterized in that The proinflammatory cytokines in the proinflammatory cytokine storm are TNF-α and / or IL-6.

8. The use according to any one of claims 5 to 7, characterized in that: The inflammatory cell infiltration is monocyte / macrophage, lymphocyte and neutrophil infiltration; Optionally, the macrophage infiltration is an increase in the proportion of CD68 positive cells.

9. The use according to any one of claims 1 to 8, characterized in that The following uses are included: (1) Use in the preparation of drugs for inhibiting pro-inflammatory cytokine storm; (2) Use in the preparation of drugs for inhibiting inflammatory cell infiltration.

10. A pharmaceutical preparation for preventing, alleviating, assisting in the treatment or treating lung injury, characterized in that: include: The active ingredient is a compound of formula I as described in any one of claims 1 to 9, its cis-trans isomers, its enantiomers, its diastereomers, its racemates, its solvates, its hydrates, or its pharmaceutically acceptable salts or prodrugs thereof; and Pharmaceutically acceptable pharmaceutical excipients or carriers.

11. The pharmaceutical preparation for preventing, alleviating, assisting in the treatment or treating lung injury according to claim 10, characterized in that: The active ingredients also include ceftazidime; Optionally, the pharmaceutical preparation is in the form of a liquid preparation and a solid preparation.

12. A method for preventing, alleviating, assisting in the treatment or treating lung injury, characterized in that: Administering the compound of formula I, its cis-trans isomers, its enantiomers, its diastereomers, its racemates, its solvates, its hydrates, its pharmaceutically acceptable salts or its prodrugs to a subject in need; R is H, fluoro or cyano; X is N or CR1, and R1 is H.

13. The method according to claim 12, characterized in that The compound represented by formula I is:

14. The method according to claim 12 or 13, characterized in that The compound represented by formula I, its cis-trans isomers, its enantiomers, its diastereomers, its racemates, its solvates, its hydrates, its pharmaceutically acceptable salts or its prodrugs are used in combination with ceftazidime.

15. The method according to any one of claims 12 to 14, characterized in that: The lung injury includes: acute lung injury or its combined diseases; or acute respiratory distress syndrome or its combined diseases; or chronic obstructive pulmonary disease or its combined diseases; Optionally, the combined disease is diabetes; Optionally, the diabetes is type 1 diabetes.

16. The method according to any one of claims 12 to 15, characterized in that: The lung injury is caused by infection, pro-inflammatory cytokine storm and / or inflammatory cell infiltration.

17. The method according to claim 16, characterized in that The infections include microbial infections; Optionally, the microbial infection includes viral infection or bacterial infection; Optionally, the viral infection is a coronavirus infection; Optionally, the coronavirus infection is SARS-CoV-2 virus infection.

18. The method according to claim 16 or 17, characterized in that The proinflammatory cytokines in the proinflammatory cytokine storm are TNF-α and / or IL-6.

19. The method according to any one of claims 16 to 18, characterized in that: The inflammatory cell infiltration is monocyte / macrophage, lymphocyte and neutrophil infiltration; Optionally, the macrophage infiltration is an increase in the proportion of CD68 positive cells.

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