Application of compound in preparation of medicine for preventing, relieving, adjunctively treating or treating lung injury

Abstract

The invention provides an application of a compound as shown in a formula I, cis-trans isomers, enantiomers, diastereoisomers, racemes, solvates, hydrates or pharmaceutically acceptable salts or prodrugs of the compound in preparation of drugs for prevention, alleviation, adjuvant therapy or treatment of lung injury, and application of the compound in preparation of drugs for prevention, alleviation, adjuvant therapy or treatment of lung injury in preparation of drugs for prevention, alleviation, adjuvant therapy or treatment of lung injury in preparation of drugs for prevention, alleviation, adjuvant therapy or treatment of lung injury. The compound shown in the formula I can obviously reduce proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) of an acute lung injury rat model, improve infiltration of inflammatory cells (including mononuclear / macrophage, lymphocyte and neutrophil) in lung tissues, improve pathological injury of the lung tissues, improve lung functions, and improve the curative effect of the lung. The above results show that the compound represented by the formula I can be used for prevention, alleviation, adjuvant therapy or treatment of lung injury, especially acute lung injury or a combined disease thereof or acute respiratory distress syndrome or a combined disease thereof.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to use of a compound in preparing a medicine for preventing, alleviating, assisting in the treatment or treating lung injury. Background Art

[0002] COVID-19 is a serious disease caused by the novel coronavirus 2 (SARS-CoV-2). Patients with COVID-19 often experience acute respiratory distress syndrome (ARDS), which can cause severe acute respiratory failure and lead to extremely high mortality. Patients with chronic diseases such as diabetes or cardiovascular disease are more likely to develop severe illness or even die after being infected with SARS-CoV-2.

[0003] Studies have shown that excessive production of proinflammatory cytokines is one of the main factors in the development of ARDS. Therefore, blocking the production of proinflammatory cytokines may be beneficial in preventing COVID-19 patients from developing ARDS. In addition, studies have found that autopsy of lung tissue of fatal COVID-19 patients showed strong infiltration of macrophages, and controlling the excessive inflammatory response produced by macrophages may have a beneficial effect on the development of ARDS in COVID-19 patients. Summary of the invention

[0004] Therefore, the technical problem to be solved by the present invention is the use of a compound in the preparation of a drug for preventing, alleviating, assisting in the treatment or treating lung injury.

[0005] To this end, the present invention provides the following technical solutions:

[0006] 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 its prodrugs 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;

[0007]

[0008] R is H, fluoro or cyano;

[0009] X is N or CR1, and R1 is H.

[0010] Optionally, the compound represented by formula I is:

[0011]

[0012]

[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 its prodrugs in combination with ceftazidime in the preparation of a drug for preventing, alleviating, assisting in the treatment or treating lung injury or in the prevention, 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 increase in the 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 treating 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 its prodrugs; 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] The technical solution of the present invention has the following advantages:

[0035] 1. The present invention 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 drugs for preventing, alleviating, adjuvant therapy or treating lung injury. The present invention has verified 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 rat models of acute lung injury, inhibit lipoprotein-associated phospholipase A2 (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 acute lung injury combined with disease (diabetes). The above shows that the compound of formula I can be used for preventing, alleviating, adjuvant therapy or treating lung injury, especially acute lung injury or its combined disease (diabetes) or acute respiratory distress syndrome or its combined disease (diabetes). BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1 It is the experimental flow chart of Experimental Example 1 of the present invention;

[0038] Figure 2is the Lp-PLA2 activity in the plasma of rats in different treatment groups in Experimental Example 1 of the present invention. All experimental data are expressed as mean ± standard error, and compared with vehicle, * represents p < 0.05, *** represents p < 0.001;

[0039] Figure 3 These are the results of cytokine determination in rat BALF in Experimental Example 1 of the present invention. All experimental data are expressed as mean ± standard error, and compared with vehicle, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001;

[0040] Figure 4 It is the experimental flow chart of Experimental Example 2 of the present invention;

[0041] Figure 5 This is the effect of SR19 on the survival rate of ALI rats with diabetes induced by different doses of LPS in Experimental Example 2 of the present invention; in the figure, V represents Vehicle, and SR represents SR19;

[0042] Figure 6 is the Lp-PLA2 activity in the plasma of SD rats in different treatment groups in Experimental Example 2 of the present invention. All experimental data are expressed as mean ± standard error, * represents p < 0.05, *** represents p < 0.001;

[0043] Figure 7 The results of rat lung tissue macrophage infiltration assay; all experimental data are expressed as mean ± standard error. In the figure, ***, **** represent p < 0.001, p < 0.0001, respectively;

[0044] Figure 8 is the experimental flow chart in Experimental Example 3;

[0045] Fig. 9 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 (Ordinary one-way ANOVA) was used for multiple comparisons using Dunnett's method. Compared with vehicle, * represents p < 0.05, *** represents p < 0.001, and "ns" means: not statistically.

[0046] Fig.10 Chemokine levels and inflammatory cell levels in BALF in Experiment 3; All experimental data are expressed as mean ± standard error, and one-way ANOVA (Ordinary one-way ANOVA) was performed using Dunnett's method for multiple comparisons. Compared with vehicle, * represents p < 0.05, *** represents p < 0.001;

[0047] Fig.11 Pathological injury score 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 -score SR15或CAZ或SR15 / CAZ ) / score vehicle *100 or improvement rate (%) = (score CAZ -score SR15 / CAZ ) / score vehicle *100;

[0048] Fig.12 is the experimental flow chart in Experimental Example 4;

[0049] Fig.13 is the level 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, one-way ANOVA (Ordinary one-way ANOVA), Dunnett's method was used for multiple comparisons, compared with Day0, * represents p < 0.05, *** represents p < 0.001;

[0050] Fig.14 is the result of monocyte counting in BALF in Experimental Example 4; all experimental data are expressed as mean ± standard error, inhibition rate (%) = (count [vehicle] -count [SR19] ) / count [vehicle] *100;

[0051] Fig.15 is the level of LysoPC detected in BALF in Experimental Example 4; all experimental data are expressed as mean ± standard error, inhibition rate (%) = (count [vehicle] -count [SR19] ) / count [vehicle] *100; Dunnett's method was used for multiple comparisons, compared with "Vehicle", * represents p<0.05, #<0.01 Remarks: The control sample is the test sample added when testing LysoPC;

[0052] Fig.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.

[0053] Fig.17 This is the flow chart of the experimental design for time-dependent efficacy evaluation in Experimental Example 5.

[0054] Fig.18This is the flow chart of the experimental design for dose-dependent efficacy evaluation in Experimental Example 5.

[0055] Fig.19 is 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.

[0056] Fig. 20 is the white blood cell count 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.

[0057] Fig.21 It is the classification count 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.

[0058] Fig. 22 is 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.

[0059] Fig.23 This is the pathological analysis of lung tissue HE staining in Experiment 5; all experimental data are expressed as mean ± standard error, two-way ANOVA (Ordinary one-way ANOVA), 2way ANOVA method was used for multiple comparisons, compared with vehicle, * represents p < 0.05, ** represents p < 0.01, ns represents no significance. The drug administration group was 10 mg / kg SR13. DETAILED DESCRIPTION

[0060] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0061] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0062] The following compounds:

[0063]

[0065] The compounds represented by the above formula I(1), formula I(2) and formula I(3) are known compounds, which 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(2) is numbered SR19, and the compound represented by formula I(3) is numbered SR15.

[0066] Experimental Example 1: Efficacy of SR13 on LPS intratracheal instillation-induced acute lung injury model in rats

[0067] 1. Experimental purpose: Previous studies have shown that the LPS-induced acute lung injury (ALI) model in rats can simulate the cytokine signaling pathways and lung tissue damage 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, this example constructed an LPS tracheal instillation-induced acute lung injury model in rats to investigate the efficacy of SR13 in acute lung injury and ARDS.

[0068] 2. Experimental design:

[0069] 1) SR13 efficacy in animals

[0070] Adult healthy male SD rats (280-330g, 6-8 weeks old) were instilled with lipopolysaccharide (LPS) through the trachea to induce an acute lung injury rat model (ALI). ALI rats were randomly divided into three drug-administered groups (SR13 doses of 1 mg / kg, 3 mg / kg and 10 mg / kg, respectively) and a vehicle group (vehicle), and sham-operated rats of the same age were used as the control group (sham). SR13 and the vehicle were administered by single oral gavage per day for 2 consecutive days, and the control group was instilled with an equal amount of normal saline through the trachea, as shown in Table 1. After administration on the second day, LPS tracheal instillation surgery was performed. Plasma and bronchoalveolar lavage fluid (BALF) were collected 6 hours after the operation, and the Lp-PLA2 activity in plasma was detected by enzymatic detection using thio-PAF as a substrate, and the cytokine levels in BALF were detected by enzyme-linked immunosorbent assay. The experimental process is shown in Figure 1 .

[0071] 2) Inhibition of in vitro activity of Lp-PLA2 in plasma of healthy and ALI rats by SR13

[0072] Plasma from healthy and ALI rats was obtained, 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.

[0073] Table 1. Animal groups and dosing regimens for the in vivo efficacy experiment of SR13

[0074]

[0075] Note: PO is oral administration; QD is once a day.

[0076] 3. Experimental preparation

[0077] 3.1 Test product information

[0078] Table 2

[0079] Test article number batch Molecular weight (salt-containing) purity(%) Storage conditions SR13 N0270-83A 490.42 99.19 4℃

[0080] 3.2 Reagent information

[0081] Table 3

[0082]

[0083]

[0084] 3.3 Main instruments and equipment

[0085] Table 4

[0086]

[0087] 3.4 Animals and breeding environment

[0088] Male adult SD rats weighing 280-330 g and aged 6-8 weeks were used in this experiment. They were provided by Zhejiang Weitong Lihua Experimental Animal Co., Ltd. The animals were kept in standard polypropylene cages. The temperature in the cage was kept at 22-24°C and the room maintained a 12-hour light / dark cycle. The animals had free access to water and food.

[0089] 3.5 SR13 efficacy in animals: experimental preparations

[0090] 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 for later use.

[0091] LPS preparation: Weigh an appropriate amount of LPS powder and dissolve it in physiological saline to prepare a solution with a concentration of 2 mg / mL. Vortex and sonicate until a clear solution is formed.

[0092] SR13 preparation: Weigh an appropriate amount of compound SR13 powder, add 1% MC solution according to the proportion, vortex and sonicate until a uniform suspension is formed.

[0093] 3.6 In vitro inhibition of Lp-PLA2 activity by SR13 in plasma of healthy and ALI rats: experimental preparation

[0094] Preparation of experimental working solution:

[0095] 1×Assay buffer solution: 100 mM Tris-base, 1 mM EGTA (adjust pH to 7.2 with HCl);

[0096] 2×Substrate solution: 0.5 mM 2-thio-PAF;

[0097] 10× DTNB solution: 10 mM DTNB.

[0098] Compound solution preparation:

[0099] SR13 solution: SR13 was prepared into a 10mM stock solution with DMSO, and then diluted 3 times with DMSO to obtain 10 consecutive compound concentrations. Then, each concentration point was diluted 50 times with 1×Assay buffer to obtain the working solution of the compound, with concentrations of 200, 66.67, 22.22, 7.41, 2.47, 0.82, 0.27, 0.09, 0.03, and 0.01μM. The final concentrations of the compound after adding it to the reaction system were: 10, 3.33, 1.11, 0.37, 0.12, 0.04, 0.01, 0.005, 0.002, and 0.0005μM.

[0100] 4. Experimental methods and steps

[0101] 4.1 SR13 efficacy in animals

[0102] 4.1.1 Establishment of rat model of acute lung injury

[0103] After the rats were anesthetized, their limbs were fixed on the surgical board, the neck was shaved and the neck skin was disinfected with iodine, the neck skin was cut, the incision was about 1 cm, the muscles were bluntly separated, and the trachea was exposed. LPS or saline was slowly and evenly injected into the trachea with a 1mL syringe with a 27-Ga needle within 30 seconds. Immediately after the injection, the rats were kept upright with their heads up and their tails down for 1 minute to allow the liquid to flow fully. The wounds were sutured after surgery. The rats undergoing surgery were placed on a heating pad to maintain body temperature until they woke up.

[0104] 4.1.2 Dosage Information

[0105] The dosing volume for each rat is implemented according to its specific body weight. The standard dosing volume for SR13 is 10 mL / kg, and the standard dosing volume for LPS is 1 mL / kg. The actual animal dosing doses corresponding to 0.1 mg / mL, 0.3 mg / mL and 1 mg / mL SR13 solutions are 1 mg / kg, 3 mg / kg and 10 mg / kg, respectively. The LPS dosing dose is 2 mg / kg. The dosing volume for each rat is based on its body weight and the concentration of the prepared SR13 suspension and LPS solution. Dosing volume calculation formula: Dosing volume (mL) = body weight (kg) × (dosage (mg / kg) / (SR13 or LPS) concentration (mg / mL)).

[0106] 4.1.3 Sample collection

[0107] 4.1.3.1 Blood sample collection

[0108] Whole blood samples were collected from all animals 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.

[0109] 4.1.3.2 BALF sample collection

[0110] At the end of the experiment, the animals were anesthetized and sacrificed by cardiac blood sampling. The chest cavity and cervical trachea were exposed by surgery. The right lung was ligated, and the left lung was lavaged twice with PBS through endotracheal intubation, and each time the BALF was collected by repeated aspiration 3 times. The recovery rate of BALF was above 80% and used for subsequent analysis.

[0111] 4.1.2 Sample determination

[0112] 4.1.4.1 Methodology for testing Lp-PLA2 activity in plasma

[0113] The determination of Lp-PLA2 activity in plasma and the inhibition test of Lp-PLA2 activity in plasma of healthy and ALI rats by SR13 were performed by using 2-thio-PAF as substrate and reading the absorbance at 405nm and 510nm wavelengths on a multifunctional microplate reader for kinetic detection. The Lp-PLA2 enzyme activity was defined as the amount of substrate hydrolyzed per milliliter of plasma per unit time (nmol), unit: nmol / min / mL.

[0114] The specific protocol for testing the activity of Lp-PLA2 in plasma obtained from the in vivo pharmacodynamics experiment is as follows: thaw the plasma at room temperature, balance it after thawing 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, item number: 655101), add 15 μL of 1×assay buffer to each well, and oscillate to mix for use; negative control well (LC): according to the 96-well assay plate, add 5 μL of 200 μM Darapladib solution (positive compound, dilute the compound with 1×Assaybuffer) to each well; for other experimental wells, according to the 96-well assay plate, add 5 μL of 2% DMSO solution (diluted with 1×assaybuffer) 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 it 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 detection plate. The detection plate was shaken and mixed and placed in an ELISA reader with the temperature set at 37°C. Kinetic detection was performed at a wavelength of 405 / 510 nm.

[0115] 4.1.4.2 Cytokine test in BALF: ELISA was used to detect the levels of TNF-α and IL-6 cytokines in BALF. The detection was performed at 450nm and 570nm wavelengths using a multifunctional microplate reader.

[0116] 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:

[0117] 1) Thaw the plasma of healthy rats and ALI rats at room temperature, balance and place in a centrifuge after thawing, centrifuge at 2000 rpm for 1 minute;

[0118] 2) 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 well for later use;

[0119] 3) The compound was serially diluted 3-fold in DMSO solution to 10 concentration points;

[0120] 4) The diluted compound was diluted 50 times with 1×Assay buffer, and 5 μL of compound dilution was added to each well of the 96-well assay plate;

[0121] 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;

[0122] Negative control wells (LC): Add 5 μL of 200 μM Darapladib solution (positive compound, dilute the compound with 1× Assay buffer) to the 96-well assay plate according to the plate layout;

[0123] 5) Add 10 μL of 10× DTNB suspension to the test plate, shake and mix until the solution is clear, and incubate at room temperature for 30 minutes;

[0124] 6) After incubation, place in a 37°C biochemical incubator for 5-10 minutes, and add 50 μL of 2× substrate solution to the test plate to start the reaction;

[0125] 7) The detection plate was shaken and mixed and placed in an ELISA reader with the temperature set to 37°C. Kinetic detection was performed at wavelengths of 405 and 510 nm.

[0126] 4.3 Statistical analysis

[0127] 4.3.1 SR13 efficacy in animals:

[0128] 4.3.1.1 Analysis of Lp-PLA2 activity in plasma. The experimental data were collected using Biotek Synergy H1, Software Version Gen 5 software, and analyzed and processed using Microsoft Excel 2010. One-way ANOVA and Dunnett's multiple comparisons test were used to analyze the differences between the groups. The statistical results were expressed as mean ± standard error, and p < 0.05 indicated that the statistical results were significantly different. SR13 inhibited Lp-PLA2 activity in the plasma of healthy and ALI rats. 50 The values ​​were analyzed by nonlinear regression using the Software Version Gen 5 software provided with the Biotek microplate reader.

[0129] 4.3.1.2 Analysis of cytokines in BALF was performed using Microsoft Excel 2010. One-way ANOVA and Dunnett's multiple comparisons test were used to analyze the differences between the groups. The statistical results were expressed as mean ± standard error, and p < 0.05 indicated that the statistical results were significantly different.

[0130] 4.3.2 In vitro inhibition of Lp-PLA2 activity by SR13 in plasma of healthy and ALI rats

[0131] In the experiment, the experimental control conditions were defined based on the signal, with the average reaction rate of Lp-PLA2 enzyme in the wells containing plasma and 0.1% DMSO as the positive control with no inhibition (defined as: High control); the average reaction rate of Lp-PLA2 enzyme in the wells containing plasma and 10μM Darapladib was used as the negative control with 100% inhibition (defined as: Low control). Inhibition rate calculation 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, data are expressed as mean ± standard deviation. Nonlinear regression analysis was performed using Software Version Gen 5 software.

[0132] 5 Experimental results and discussion

[0133] 5.1 SR13 efficacy in animals:

[0134] 5.1.1 Results of Lp-PLA2 activity determination in plasma

[0135] The results of the plasma Lp-PLA2 activity test showed that the plasma Lp-PLA2 activity in ALI rats was significantly increased (p<0.05) ( Figure 2 ). The Lp-PLA2 activity in the plasma of rats in the 1, 3, and 10 mg / kg / day SR13 administration groups decreased by 76.39±1.98% (mean±SE, n=9), 91.19±1.38% (mean±SE, n=9), and 99.47±0.38% (mean±SE, n=9), respectively, compared with the vehicle group (vehicle), showing a dose-dependent decrease. The results are shown in Table 5. Figure 2 , in the figure, mpk = mg / kg.

[0136] Table 5. SR13 inhibits Lp-PLA2 activity in plasma of ALI rats

[0137]

[0138] 5.1.2 Cytokine test results in BALF

[0139] In the ALI rat model, TNF-α and IL-6 levels in BALF were significantly increased after 6 h of LPS stimulation (p<0.001) ( Figure 3 ). Compared with the vehicle, the TNF-α in BALF of the 1 mg / kg, 3 mg / kg and 10 mg / kg SR13 administration groups decreased by 28.93±12.24% (mean±SE, n=14), 34.03±10.37% (mean±SE, n=14) and 47.23±6.82% (mean±SE, n=14), respectively; the IL-6 in BALF decreased by 20.03±19.95% (mean±SE, n=14), 7.49±15.89% (mean±SE, n=14) and 54.91±5.61% (mean±SE, n=14), respectively. SR13 inhibited the increase of BALF cytokines in a dose-dependent manner.

[0140] (Table 6, Figure 3 )

[0141] Table 6. SR13 inhibits the increase of cytokines in BALF

[0142]

[0143]

[0144] In summary, 6 hours after LPS modeling, the Lp-PLA2 activity in plasma (p<0.05) and the levels of TNF-α and IL-6 in bronchoalveolar lavage fluid (BALF) (p<0.001) of ALI rats in the vehicle group (LPS+vehicle) were significantly increased. SR13 had a dose-dependent inhibitory effect on the increase of Lp-PLA2 enzyme activity in plasma and TNF-α and IL-6 in BALF.

[0145] 5.2 Inhibition of in vitro activity of Lp-PLA2 in plasma of healthy and ALI rats by SR13

[0146] As shown in Tables 7 and 8 below, SR13 inhibited 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.

[0147] Table 7. Summary of the inhibitory data of SR13 on Lp-PLA2 activity in plasma of healthy SD rats

[0148]

[0149] Table 8. Summary of the data on the inhibition of Lp-PLA2 activity in plasma of ALI rats by SR13

[0150]

[0151] Experimental Example 2: Efficacy of SR19 on LPS intratracheal instillation-induced acute lung injury model in rats

[0152] 1. Experimental purpose: Previous studies have shown that the ALI rat model induced by LPS tracheal instillation can simulate cytokine signaling pathways and lung tissue damage similar to ARDS in COVID-19 patients. This experiment used type 1 diabetic rats combined with ALI modeling to simulate COVID-19-related ARDS on the basis of diabetes. This study aims to evaluate the efficacy of SR19 on the rat acute lung injury model induced by LPS tracheal instillation in type 1 diabetic rats.

[0153] 2. Experimental design:

[0154] 1) SR19 efficacy in animals

[0155] After the rats arrived at the facility and adapted to the environment for 2-3 days, STZ solution (60 mg / kg) was injected intraperitoneally to induce type I diabetes. One week after STZ injection, rats with blood glucose values ​​greater than 16.7 mmol / L were screened for LPS-induced acute lung injury modeling.

[0156] The animals were divided into 7 experimental groups, namely Naive (normal rats), Naive+Vehicle, Naive+SR19, LPS+Vehicle, LPS+SR19, STZ+LPS+Vehicle, and STZ+LPS+SR19. The dose of SR19 was 10 mg / kg. SR19 and the vehicle were administered by oral gavage once a day starting one day before LPS induction for 3 consecutive days (see Figure 4 The rats were observed after LPS surgery and the mortality rate was recorded. The experimental endpoint was reached 48 hours after LPS induction, and the animal lung tissues and plasma were collected for subsequent analysis.

[0157] The lung tissue macrophage infiltration test is a single-blind test. After sampling the animal lung tissue, the lung tissue samples with only the animal number information will be handed over to Nantong WuXi AppTec Pharmaceuticals Co., Ltd. for IHC staining analysis. After Nantong WuXi AppTec Pharmaceuticals Co., Ltd. has fed back all the original data, the test will be unblinded for data analysis.

[0158] 2) SR19 inhibits the in vitro activity of Lp-PLA2 in the plasma of healthy SD rats

[0159] The plasma of healthy SD rats was obtained, 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 SD rats.

[0160] 3. Experimental preparation

[0161] 3.1 Test product information

[0162] Table 9

[0163] Test article number batch Molecular weight (salt-containing) purity(%) Storage conditions SR19 N0419-06A 496.446 98.92 4℃

[0164] 3.2 Reagent information

[0165] Table 10

[0166]

[0167] Table 11

[0168]

[0169]

[0170] 3.3 Animals and breeding environment

[0171] This experiment used male adult SD rats, weighing 240g-260g, 6 weeks old, provided by Zhejiang Weitong Lihua Experimental Animal Co., Ltd. The animals were kept in standard polypropylene cages, the temperature in the breeding room was stabilized at 22-26℃, and the room maintained a 12-hour light / dark cycle. The animals had free access to water and food.

[0172] 3.4 SR19 Animal In Vivo Efficacy Experiment Preparation

[0173] 0.1mol / L citric acid buffer: Accurately weigh 4.2g of citric acid powder into a reagent bottle, add 150ml of deionized water and stir until the solution is fully dissolved, adjust the pH to 4.4, make up to 200ml, and store in a 4℃ refrigerator for later use.

[0174] STZ solution: Weigh an appropriate amount of STZ reagent and dissolve it in 0.1 mol / L citric acid buffer, vortex and oscillate until it dissolves into a clear solution. Note: The preparation process should be kept away from light and should be used immediately after preparation and injected within 10 minutes after preparation.

[0175] 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 for later use.

[0176] 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.

[0177] LPS preparation: Weigh an appropriate amount of LPS powder and dissolve it in physiological 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.

[0178] 3.5 Experimental preparation of SR19 to inhibit in vitro activity of Lp-PLA2 in plasma of healthy SD rats

[0179] Preparation of experimental working solution:

[0180] 1×Assay buffer: 50 mM HEPES, 1 mM CHAPS, and 150 mM NaCl (pH adjusted to 7.4 with NaOH);

[0181] 5×Substrate solution: 2.5 mM 2-thio-PAF, 3.2 mM NEM, 0.032 mM CPM; Termination solution: 5% TFA.

[0182] SR19 solution preparation:

[0183] SR19 was prepared into a 10mM stock solution with DMSO. In the experiment, the 10mM SR19 stock solution was diluted with DMSO to a 1mM secondary stock solution, and then 3-fold serial dilutions were performed with DMSO to obtain 10 consecutive compound concentrations. After that, each concentration point was diluted 20-fold with 1×Assay buffer to obtain the working solution of the compound, with concentrations of 50, 16.67, 5.56, 1.85, 0.62, 0.21, 0.07, 0.02, 0.008, 0.003μM. The final concentrations of the compound after adding it to the reaction system were: 1, 0.33, 0.11, 0.04, 0.01, 0.004, 0.001, 0.0005, 0.0002, 0.00005μM.

[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] After the rats arrived at the animal facility, they adapted to the living environment for 2-3 days, and the rats' body weight and basal blood sugar level after 6 hours of fasting were measured. Subsequently, STZ induction was performed, and the rats were intraperitoneally injected with 60 mg / kg STZ solution after 6 hours of fasting, and then given 10% glucose water for 24 hours to prevent the animals from hypoglycemia and shock. The fasting blood sugar of the rats was measured on the 7th day after STZ injection. Rats with blood sugar values ​​greater than 16.7 mmol / L were type I diabetic rats, and subsequent LPS induction was performed.

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

[0189] After the rats were anesthetized, their limbs were fixed on the surgical board, the neck was shaved and the neck skin was disinfected with iodine, the neck skin was cut, the incision was about 1 cm, the muscles were bluntly separated, and the trachea was exposed. The LPS preparation or saline was slowly and evenly injected into the trachea with a 1mL syringe with a 27-Ga needle within 30 seconds. Immediately after the injection, the rat was kept upright with its head up and its tail down for 1 minute to allow the liquid to flow fully. The wound was sutured after surgery. The rats undergoing surgery were placed on a heating pad to maintain body temperature until they woke up.

[0190] 4.1.3 Dosage information

[0191] The dosage volume of each rat was implemented according to its specific body weight, dosage and reagent concentration. The dosage of STZ was 60 mg / kg and that of SR19 was 10 mg / kg. The actual dosage of animals corresponding to 1 mg / mL, 2 mg / mL and 5 mg / mL LPS solutions were 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, the mortality rate of the animals was recorded. Animals that survived 48 hours after LPS modeling were anesthetized and sacrificed by cardiac blood sampling. Whole blood samples were collected in centrifuge tubes containing K2EDTA, centrifuged at 5000 rpm for 10 minutes (4°C) to separate plasma, and then stored in a -80°C refrigerator for analysis.

[0196] 4.1.4.2 Lung tissue samples

[0197] At the end of the experiment, the animals were anesthetized and sacrificed by cardiac blood sampling, and the chest cavity and cervical trachea were exposed by surgery. The right lung was ligated, and the left lung was fixed with 0.2 mL 4% PFA through endotracheal intubation. After ligation, it was placed in a tissue embedding box, completely immersed in sufficient 4% PFA, and stored at 4°C for subsequent detection and 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 time. 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, their 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 test method for Lp-PLA2 activity in plasma uses 2-thio-PAF as the substrate and reads the absorbance value at 405nm and 510nm wavelengths on a multifunctional microplate reader for kinetic detection. The Lp-PLA2 enzyme activity is defined as the amount of substrate hydrolyzed per milliliter of plasma per unit time (nmol), unit: nmol / min / mL.

[0203] The specific method is as follows: thaw the plasma at room temperature, balance it and place it in a centrifuge after thawing, centrifuge it at 2000 rpm for 1 minute; add 20 μL of plasma to a 96-well test plate (brand: Greiner, item number: 655101), add 15 μL of 1×assay buffer to each well, oscillate and mix well for use; negative control well (LC): according to the platemap, 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 platemap, 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 and 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 detection plate. The detection plate was shaken and mixed and placed in an ELISA reader with the temperature set at 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] The lung tissue was cut into 4 μm paraffin sections in the direction of the maximum cross section. The sections were dewaxed, dehydrated, and sealed with a membrane, stained with recombinant CD68 antibodies, sealed, and scanned and collected using a high-throughput digital slide scanner. The proportion of CD68-positive cells to total cells in the lung tissue sample of each animal was counted 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 after thawing, centrifuge it at 2000 rpm for 1 minute;

[0208] 2) Add 40 μL of plasma to each well of a 96-well assay plate (brand: Greiner, catalog number: 655076) for later use;

[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 mixed, 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) The 96-well test plate was leveled and placed in a centrifuge for centrifugation 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 the mortality rate of ALI rats. The experimental data were analyzed and processed using Microsoft Excel 2010.

[0219] 4.3.1.2 Analysis of Lp-PLA2 activity in plasma. The experimental data were collected using Biotek Synergy H1, Software Version Gen 5 software, and analyzed and processed using Microsoft Excel 2010. One-way ANOVA and Tukey's multiple comparisons test were used to analyze the differences between the groups. The statistical results were expressed as mean ± standard error, and p < 0.05 indicated that the statistical results were significantly different. SR19 inhibited 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 the data of macrophage infiltration in lung tissue, the animal grouping information was masked, and the number of all cells and CD68-positive cells in the lung tissue sections of each animal were counted. The total number of CD68-positive cells in the lung tissue of each animal was divided by the total number of cells, which was the percentage of CD68-positive cells in the lung tissue of each animal, representing the macrophage infiltration of the lung tissue of each animal. All data were imported into Microsoft Excel 2010 to analyze the percentage of CD68-positive cells in the lung tissue of each animal, and then the grouping information was unblinded, and a two-way ANOVA was performed, and the Tukey method was used for multiple comparisons. The statistical results were expressed as mean ± standard error, and p < 0.05 indicated that the statistical results were significantly different.

[0221] 4.3.2 Inhibition of in vitro activity of SR19 on Lp-PLA2 in plasma of healthy rats

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

[0223] In the experiment, the experimental control conditions were defined based on the signal. The average value of Lp-PLA2 enzyme activity in the wells containing plasma and 0.1% DMSO was used as the positive control with no inhibition (defined as: High control); the average value of Lp-PLA2 enzyme activity in the wells containing plasma and saturated concentration of standard compound was used as the negative control with 100% inhibition (defined as: Low control). Inhibition rate calculation 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 in each experiment was n ≥ 3, the data were expressed as mean ± standard deviation (SD); if n < 3, the data were expressed as 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] The animals were given 10 mg / kg SR19 by gavage one day before LPS tracheal instillation for 3 consecutive days and the survival rate of the rats was continuously monitored for 48 hours after LPS tracheal instillation. The results showed that under the condition of 5 mg / kg LPS modeling dose, ALI modeling caused more animals to be dying and die in type I diabetic rats. Continuous gavage of 10 mg / kg SR19 for 3 consecutive days had a protective effect on the mortality of ALI rats with diabetes (see Table 12, Figure 5), but had a weak protective effect on LPS-induced 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 in the dying state appeared in all groups; 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 the dying state, and 20% of the rats were in the normal state), and 100% in the STZ+LPS+SR19 group. In Table 12, the number of animals in each group at a dose of 1 mg / kg of LPS (including Naive+Vehicle and Naive+SR19 groups) was 7, the number of animals in groups other than LPS+SR19 at a dose of 2 mg / kg (including Naive group) was 9, and the number of animals in LPS+SR19 was 10. At a dose of 5 mg / kg, 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 rate of diabetic rats and normal rats induced by different doses of LPS

[0229]

[0230] 5.1.2 Results of Lp-PLA2 enzyme activity in plasma

[0231] The results of the plasma Lp-PLA2 activity test showed that the Lp-PLA2 activity in the plasma of ALI rats induced by 1 mg / kg LPS (p<0.05) and the Lp-PLA2 activity in the plasma of ALI rats induced by STZ+LPS (p<0.05) were significantly increased ( Figure 6 ). In the ALI rat models induced by 1 mg / kg LPS and STZ+LPS, the Lp-PLA2 activity in the plasma of rats in the 10 mg / kg / day SR19 administration group decreased by 99.48±0.31% (mean±SE, n=7) and 94.42±2.27% (mean±SE, n=7) respectively compared with the corresponding vehicle group (Vehicle). The results are shown in Table 13.

[0232] Table 13. SR19 inhibits Lp-PLA2 enzyme activity in SD rat plasma

[0233]

[0234] 5.1.3 Analysis of macrophage infiltration in lung tissue

[0235] In lung tissue, CD68 antibody can be used to mark activated and polarized macrophages. In this study, the quantitative analysis method of CD68 antibody IHC staining results was used to explore the macrophage infiltration in lung tissue. 48 hours after LPS intratracheal instillation, the results showed that the lung tissues of diabetic rats and normal rats induced by 1 mg / kg LPS showed obvious macrophage infiltration, and continuous oral administration of 10 mg / kg SR19 for 3 consecutive days had a certain inhibitory effect on the macrophage infiltration in the lung tissues of ALI rats with diabetes (see Table 14, Figure 7 ), but had a weak protective effect on LPS-induced normal rats (see Table 14, Figure 7 ). The percentages of CD68 positive cells in the lung tissues of ALI rats treated with SR19 were: The percentage of CD68 positive cells in the lung tissues of ALI rats without medication was: The difference between the two groups was 8.7084±0.638% (mean±standard error, n=5), the difference between the two groups was 15.9792±1.379% (mean±standard error, n=5), and the difference between the two groups was 17.2754±1.149% (mean±standard error, n=5). In comparison, 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% (mean±SE, n=5), respectively; compared with ALI diabetic rats not treated with the drug, the proportion of CD68-positive cells in the lung tissues of ALI diabetic rats treated with SR19 decreased by 24.45±7.37% (mean±SE, n=5), p=0.06.

[0236] Table 14. Percentage of CD68 positive cells in rat lung tissue

[0237]

[0238]

[0239] 5.2 Inhibition of in vitro activity of Lp-PLA2 in plasma of healthy SD rats by SR19

[0240] 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 It is 12.25 nM (average value, n=2).

[0241] Table 15. Summary of the data on the inhibition of Lp-PLA2 activity in plasma of healthy SD rats by SR19

[0242] 1st time 2nd time average value Bottom 8.46 -16.61 -4.08 Top 99.94 88.44 94.19 HillSlope 2.14 1.39 1.76 <![CDATA[IC 50 (nM)]]> 13.07 11.42 12.25

[0243] In summary, this experimental example compared the harmful effects of lipopolysaccharide (LPS) tracheal instillation on normal rats and type 1 diabetic rats after acute lung injury (ALI), and on this basis evaluated the protective effect of SR19 on these two ALI rats. The results showed that ALI modeling caused a higher mortality rate in type 1 diabetic rats, and SR19 could 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 10 mg / 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).

[0244] Experimental Example 3: Study on the efficacy of SR15 on the mouse model of acute pneumonia induced by nasal instillation of Pseudomonas aeruginosa

[0245] 1. Introduction

[0246] Pneumonia is a common acute respiratory infection, which is roughly divided into community-acquired pneumonia and hospital-acquired pneumonia. The development of the disease depends largely on the host's immune response. The excessive production of proinflammatory cytokines is one of the main factors that accelerate the development of pneumonia into severe illness or acute respiratory distress syndrome (ARDS). Therefore, timely blocking the production of proinflammatory cytokines may help prevent pneumonia patients from developing severe illness or ARDS.

[0247] 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 PLA2G7high macrophages in pulmonary fibrosis.PharmacolRes.2022Aug;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 biologically active 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.2020Jan;40(1):79-134.).

[0248] Pseudomonas aeruginosa (PA) is a Gram-negative bacterium that secretes a variety of virulence factors, including endotoxins (lipopolysaccharides), exotoxins and elastase, and is widely present in nature. The PA-induced acute pneumonia mouse model well simulates the physiological processes of inflammation and lung injury after acute pneumonia, has clinical relevance, and is 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 in chronic lung disease and Pseudomonas aeruginosa-associated populations is 67% (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 hospitalised 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 nasal instillation of Pseudomonas aeruginosa.

[0249] 2. Experimental Design

[0250] Experimental design Figure 8 As shown, after the experimental mice (C57BL / 6 mice, 8-10w, male) arrived at the facility and adapted to the environment for 1 week, the experimental animals were divided into 5 groups, namely Control, PA mice+vehicle, PA mice+10mg / kg SR15, PA mice+50mg / kg CAZ (Ceftazidime), and PA mice+10mg / kg SR15 / 50mg / kg CAZ.

[0251] 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 dripped into the nasal cavity 1 hour after the first administration. PA (1×10 7CFU, ATCC 27853) were induced for 48 hours and then the mouse plasma, bronchoalveolar lavage fluid (BALF) and lung tissue were collected. The activity of Lp-PLA2 in plasma and the levels of cytokines and inflammatory cells in BALF were detected, and the lung tissue was pathologically analyzed. There is no antibacterial or bactericidal effect in the mechanism of action of Lp-PLA2 inhibitors, so the drug efficacy was evaluated in combination with antibiotics (CAZ). The antibiotics were given twice a day (12h+12h) 12 hours after PA induction. The activity of Lp-PLA2 in plasma was detected by enzymatic method using thio-PAF as substrate, the level of cytokines in BALF was detected by enzyme-linked immunosorbent assay, the classification and count of inflammatory cells in BALF was detected by blood analyzer, and the lung tissue was subjected to single-blind lung tissue pathological analysis by hematoxylin-eosin (HE) staining.

[0252] 3. Experimental results

[0253] 3.1. SR15 significantly inhibited the increase of Lp-PLA2 activity in plasma of mice with acute pneumonia induced by PA

[0254] In the PA-induced acute pneumonia mouse model, the activity of Lp-PLA2 in plasma increased significantly. CAZ had no effect on the activity of Lp-PLA2 in plasma, and the inhibition rate of SR15 or SR15 / CAZ on the activity of Lp-PLA2 in plasma was greater than 90%. Fig. 9 and Table 16.

[0255] Table 16. Inhibitory effect of SR15 on Lp-PLA2 activity in plasma of mice with acute pneumonia induced by PA

[0256]

[0257] 3.2. SR15 inhibits the secretion of chemokines and infiltration of inflammatory cells in BALF of mice with acute pneumonia induced by PA

[0258] Results Fig.10 As shown, the level of monocyte chemoattractant protein (MCP-1) in BALF of mice with PA-induced acute pneumonia 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 alone showed an inhibitory effect on neutrophils in BALF. 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 that of CAZ alone.

[0259] 3.3. SR15 improves lung tissue pathological damage in mice with acute pneumonia induced by PA

[0260] After mice were infected with PA, obvious pathological damage was found in the lung tissue. The severity of the damage was scored from multiple dimensions. The scoring criteria for each dimension in the HE score were: 0, absent; 1, minor; 2, major. Fig.11 The results showed that in the evaluation index of interstitial and alveolar hemorrhage, the improvement rate of SR15 alone was 40%, the improvement rate of CAZ alone was 66.6%, and the combined use was 66.6%; in the evaluation index of perivascular edema, the improvement rate of SR15 and CAZ alone was 16.7%, and the improvement rate increased to 44.4% after combined use; in the evaluation index of interstitial inflammation, the improvement rate of SR15 alone was 33.3%, and CAZ alone showed no improvement. Compared with CAZ alone, the improvement rate after combined use was 37.5%; in the evaluation index of perivascular inflammation, the improvement rate of SR1 alone was 20%, and CAZ alone showed no improvement. Compared with CAZ alone, the improvement rate of combined use was 50%. The results suggest that SR1 has a certain improvement effect on lung tissue pathological damage. At the same time, it can be used in combination with antibiotics to further enhance the improvement of lung tissue pathological damage.

[0261] In summary, in the microbial (Pseudomonas aeruginosa)-induced acute pneumonia mouse model, the activity of Lp-PLA2 in plasma was significantly increased. For the first time, it was demonstrated in the bacterial microbial-induced acute pneumonia model that Lp-PLA2 inhibitor (SR1) can effectively inhibit the secretion of chemokines in the BALF of pneumonia mice and inhibit the infiltration of inflammatory cells in the BALF. The drug alone has a certain improvement effect on lung tissue pathological damage, and combined with antibiotics, it further enhances the improvement of lung tissue pathological damage.

[0262] Experimental Example 4: Study on the efficacy of SR19 on the acute lung injury model of COPD rats induced by smoke combined with LPS

[0263] 1. Introduction

[0264] Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory disease characterized by persistent and progressive airflow limitation.

[0265] 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 biologically active 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.).

[0266] The rat model of acute lung injury in COPD induced by cigarette smoke combined with lipopolysaccharide (LPS) showed pathological changes such as bronchial mucosal epithelial damage at all levels, increased airway secretions, airway airflow obstruction, and inflammatory cell infiltration in the airway 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 acute lung injury model of COPD rats induced by cigarette smoke combined with LPS.

[0267] 2. Experimental Design

[0268] 2.1 Experimental animals and groups

[0269] After the experimental rats (SD rats, 8-10 weeks, male) arrived at the facility and adapted to the environment for 1 week, they were divided into two groups, namely the model control group (Vehicle) and the model administration group (SR19).

[0270] 2.2 Experimental process of smoke fumigation combined with LPS airway atomization

[0271] The experimental process is shown in Fig.12 On Day 0, after the lung function Penh test, ~500 μl of whole blood was collected from the vein and placed in an EP tube containing the anticoagulant EDTA·K2. Then, it was centrifuged at 3500 rpm, 4°C, for 10 min to prepare 100 μl*2 tubes of plasma. The prepared plasma was frozen at -80°C for later use.

[0272] From Day 1 to Day 38, rats were exposed to cigarette smoke (cigarettes: Huangguoshu, manufacturer: Guizhou China Tobacco Industry Co., Ltd.; tar content: 10 mg; smoke nicotine content: 0.9 mg; smoke carbon monoxide content: 12 mg), twice a day, 1 hour each in the morning and afternoon, 20 cigarettes each time; blood was collected on Day 7 and Day 14 for the detection of Lp-PLA2 and oxLDL levels. Before smoking on Day 10 and Day 25, LPS was injected into the airway by aerosol (doses of 2.5 mg / kg and 2 mg / kg, respectively), the doses are shown in Fig.12 .

[0273] 2.3 Pulmonary function test

[0274] Rats were placed in the plethysmography chamber of the DSI Buxco WBP system on Day 0 (before fumigation) before smoke + LPS modeling, Day 23 (before drug administration) after modeling, Day 30 (7 days of drug administration) and Day 38 (14 days of drug administration) after modeling to detect the enhanced expiratory pause (PenH) of the animals in the awake state.

[0275] 2.4 Animal dosing regimen and endpoint sample collection

[0276] On Day 24, rats were divided into groups according to body weight, Penh and other indicators, and each group of rats was given the corresponding solvent or compound for 14 consecutive days from D24 to D38. At the end of the experiment, BALF samples were collected and tested for leukocyte classification and lysophosphatidylcholine (LysoPC).

[0277] 3. Experimental results

[0278] 3.1 Results of plasma Lp-PLA2 and oxLDL levels

[0279] 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. Fig.13 .

[0280] 3.2. Results of leukocyte count and LysoPC analysis in BALF

[0281] The results of white blood cell count showed that compared with the model control group (vehicle), the monocyte infiltration in the BALF of the 10mg / kg SR19 administration group decreased by 58.6%. Fig.14 .

[0282] 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 Raviet al. Metabolic differences between bronchial epithelium from healthy individuals and patients with asthma and the effect of bronchialthermoplasty. Clinical Trial J Allergy Clin Immunol. 2021Nov; 148(5): 1236-1248.). Results are shown in Fig.15 .

[0283] 3.3. SR19 significantly improved the lung function of rats with acute lung injury induced by smoke and LPS. The lung function of experimental rats was tested with the volumetric plethysmography system of DSI Buxco WBPsystem, and the results were expressed as the PenH index. The results showed that before the administration of SR19, the baseline levels of PenH in the model control group (Vehicle) and the SR19-administered group were basically the same (Day 23). Seven days after the administration of SR19, the PenH index of the model control group continued to increase. The SR19-administered group significantly inhibited the increase of the PenH index (P<0.01), which showed a continuous downward trend compared to before the administration. The results are shown in Fig.16 .

[0284] In summary, the plasma levels of Lp-PLA2 and its substrate oxLDL were significantly increased in the acute lung injury model of COPD rats induced by smoke combined with LPS. For the first time, it was demonstrated that SR19 can significantly inhibit the hydrolysis of oxLDL by Lp-PLA2 to produce LysoPC, a proinflammatory product of oxLDL, inhibit the infiltration of pulmonary monocytes and significantly improve lung function in the acute lung injury model of COPD rats. This is the first demonstration of the therapeutic potential of Lp-PLA2 inhibitors in the acute exacerbation of COPD patients.

[0285] Experimental Example 5: Study on the efficacy of SR13 on the acute lung injury model induced by LPS tracheal aerosol in diabetic rats

[0286] 1. Introduction

[0287] Elderly people with chronic underlying diseases are the group most affected by pneumonia. Chronic underlying diseases increase the susceptibility to pneumonia and delay its resolution; pneumonia can induce the deterioration of chronic underlying diseases, making pneumonia patients with chronic underlying diseases more likely to develop severe and critical illness [see Lee J Quinton et al. Integrative Physiology of Pneumonia. Physiol Rev. 2018 Jul 1; 98(3): 1417-1464.].

[0288] 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.].

[0289] Studies have shown that delayed lung tissue repair in LPS-induced acute lung injury rodent models under diabetic conditions can simulate clinical phenomena such as poor prognosis and longer recovery time after pneumonia events in patients with chronic underlying pneumonia [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 aims to evaluate the efficacy of SR1375A on the LPS tracheal aerosol-induced acute lung injury model in diabetic rats.

[0290] 2. Experimental Design

[0291] 2.1 Experimental design for evaluating the time-dependent efficacy of SR13

[0292] After rats (SD adult males, 6-8 weeks old) arrived at the facility and adapted to the environment for 2-3 days, STZ solution (60 mg / kg) was injected intraperitoneally to induce the diabetic model. Based on the diabetic rats, the acute lung injury model (STZ / LPS ALI) was induced by tracheal aerosol injection of LPS.

[0293] Animals were analyzed for endpoints on the third day (LPS-Day 3) and the fifth day (LPS-Day 5) after LPS induction, as shown in Table 17. Both groups of animals (STZ / LPS-Day 3 and STZ / LPS-Day 5) were divided into three experimental groups: STZ / LPS+Vehicle, STZ / LPS+SR13. The dose of SR13 was 10 mg / kg. SR13 and the vehicle were administered by gavage once a day starting one day before LPS induction for 3 and 5 consecutive days. Fig.17 At the end of the experiment, BALF and lung tissues of rats were collected for subsequent analysis.

[0294] Table 17 Animal grouping and dosing regimen for SR13 in vivo efficacy experiment

[0295]

[0296] 2.2 Experimental design for evaluating the dose-dependent efficacy of R13

[0297] 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+1mg / kg SR13, STZ / LPS+3mg / kg SR13, STZ / LPS+10mg / kg SR13. SR13 and vehicle were administered by gavage once a day starting one day before LPS induction for 5 consecutive days, see Table 18. The experimental endpoint was reached on the 5th day after single LPS induction, and plasma and BALF were collected for subsequent analysis, see Fig.18 .

[0298] Table 18. Animal groups and dosing regimens for the in vivo efficacy experiment of SR13

[0299]

[0300] In the experiment, plasma samples were tested for Lp-PLA2 enzyme activity and SR1375A concentration. Plasma samples were sent to Shanghai Jingtai Biotechnology Co., Ltd. on dry ice for plasma drug concentration testing; BALF was sampled and processed, placed in a wet ice box and sent to Shanghai Bikekoyi Biotechnology Co., Ltd. on the same day for white blood cell classification testing; lung tissue was placed in a 50mL centrifuge tube filled with 4% paraformaldehyde (Paraformaldehyde, PFA), and the centrifuge tube was placed in a foam box with an ice pack and mailed to Wuhan Saiweier Biotechnology Co., Ltd. for pathological analysis.

[0301] 3. Experimental preparation

[0302] 3.1. Test product information

[0303] Table 19. Test product information

[0304] Test article number batch Molecular weight (salt-containing) purity(%) Storage conditions SR13 N0270-86B 490.42 98.5 4℃

[0305] 3.2 Reagent Information

[0306] Table 20, Reagent Information

[0307]

[0308]

[0309] 3.3 Main instruments and equipment

[0310] Table 21. Main instruments and equipment

[0311]

[0312] 3.4 Animals and breeding environment

[0313] Male adult SD rats weighing 280-330 g were used in this experiment and were provided by Zhejiang Weitong Lihua Experimental Animal Co., Ltd. The animals were kept in standard polypropylene cages, the temperature in the cage was kept at 22-24°C, and the room maintained a 12-hour light / dark cycle. The animals were free to drink water and eat, and the cages were disinfected and replaced regularly.

[0314] 3.5 Experimental preparations

[0315] 0.1mol / L citric acid buffer: Accurately weigh 4.2g of citric acid powder into a reagent bottle, add 150ml of deionized water and stir until the solution is fully dissolved, adjust the pH to 4.4, make up to 200ml, and store in a 4℃ refrigerator for later use.

[0316] STZ solution: Weigh an appropriate amount of STZ reagent and dissolve it in 0.1 mol / L citric acid buffer, vortex and oscillate until it dissolves into a clear solution. Note: The preparation process should be kept away from light and placed on ice. It should be used immediately after preparation and injected within 10 minutes after preparation.

[0317] 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 for later use.

[0318] 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.

[0319] SR13 preparation: Weigh an appropriate amount of compound SR13 powder, add 1% MC solution according to the proportion, vortex and sonicate until a uniform suspension is formed.

[0320] 4. Experimental methods and steps

[0321] 4.1. Establishment of acute lung injury model in diabetic rats

[0322] After the rats arrived at the animal facility, they adapted to the living environment for 2-3 days, and the rats' body weight and basal blood glucose level after 6 hours of fasting were measured. Subsequently, STZ induction was performed. After the rats fasted for 6 hours, 60 mg / kg STZ solution was intraperitoneally injected, and then 10% sugar water was given to the rats for 24 hours to prevent the animals from hypoglycemia shock and death. The fasting blood glucose of the rats was measured on the 7th day after STZ injection, and rats with blood glucose values ​​greater than 11mmol / L were defined as diabetic rats. For rats with blood glucose values ​​lower than 11mmol / L, STZ was injected twice, and the fasting blood glucose value was tested again one week after the supplementary injection. The diabetic rats underwent subsequent LPS induction. After the rats were anesthetized with isoflurane, they were fixed on the intubation platform, and the atomizer needle was inserted into the trachea through the mouth through the laryngoscope. The syringe plunger was quickly pushed to deliver LPS to the lungs in the form of an aerosol. After the injection was completed, the rats were carefully observed to prevent suffocation, and they were returned to the feeding room after they woke up.

[0323] 4.2. Dosage Information

[0324] The LPS administration volume was 200 μL. The SR13 administration volume for each rat was implemented according to its specific body weight. The SR13 administration volume standard was 10 mL / kg. The actual animal administration 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 concentration of the prepared SR13 suspension and LPS solution. Administration volume calculation formula: Administration volume (mL) = body weight (kg) × (administration dose (mg / kg) / (SR13 or LPS) concentration (mg / mL)).

[0325] 4.3 Sample Collection

[0326] 4.3.1 Blood sample collection

[0327] At the end of the experiment, whole blood samples were collected in centrifuge tubes containing EDTA-K2, centrifuged at 5000 rpm for 10 min (4°C) to separate plasma, and then stored in a -80°C refrigerator for analysis.

[0328] 4.3.2 BALF supernatant samples and BALF cell collection

[0329] At the end of the experiment, the animals were anesthetized and blood was collected from the abdominal aorta before being killed. The chest cavity and cervical trachea were exposed surgically. A slipknot was tied in the left bronchus with a thread, and 2.5 mL of pre-cooled saline was injected into the right lobe with a small pressure for lavage. The alveolar lavage fluid was slowly and repeatedly aspirated twice, lavaged twice, and the two lavage fluids were combined and placed on ice. The alveolar lavage fluid was centrifuged at 4°C, with a speed of 1500r / min for 10min. After centrifugation, the BALF supernatant was carefully aspirated, transferred to a new centrifuge tube and aliquoted, 1.0 mL per tube, and stored in a -80°C refrigerator. Samples with a BALF recovery rate greater than 80% were used for subsequent experiments. Immediately after the supernatant was transferred, the cell sediment was resuspended with 500uL of saline, and the resuspended liquid was transferred to a 1.5mL centrifuge tube and stored on ice. On the same day, it was sent to Shanghai Bikeko Wing Biotechnology Co., Ltd. for leukocyte classification detection.

[0330] 4.3.3 Lung tissue collection

[0331] After taking the alveolar lavage fluid, the left lung slipknot was opened, 0.5 mL of 4% PFA tissue fixative was injected into the left lung, and then the left alveoli were 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 Saiweier Biotechnology Co., Ltd. on the same day for HE staining pathological analysis.

[0332] 4.4. Sample determination

[0333] 4.4.1. Detection of plasma SR13 concentration: Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to determine the concentration of SR13 in SD rat plasma, and the test was completed by Shanghai Jingtai Biotechnology Co., Ltd.

[0334] 4.4.2. Determination of Lp-PLA2 activity in plasma: 2-thio-PAF was used as substrate, and the absorbance was read at 405nm and 510nm on a multifunctional microplate reader for kinetic detection. Lp-PLA2 enzyme activity was defined as the amount of substrate hydrolyzed per milliliter of plasma per unit time (nmol), unit: nmol / min / mL.

[0335] 4.4.3. Albumin test in BALF: Enzyme-linked immunosorbent assay (ELISA) was used to detect albumin levels in BALF, using a multifunctional microplate reader at wavelengths of 450 nm and 510 nm.

[0336] 4.4.4. White blood cell differential count in BALF: The test was performed automatically using the XT-2000i fully automatic blood analyzer, and the test was completed by Shanghai Bikeway Biotechnology Co., Ltd.

[0337] 4.4.5. Lung tissue pathology analysis: Wuhan Saiweier Biotechnology Co., Ltd. completed the pathology analysis, which was a double-blind analysis. The lung tissue pathology scoring standard four-level grading system is as shown in Table 22:

[0338] Table 22. Four-level grading system for lung tissue HE staining pathological scoring

[0339]

[0340] [US] Peter. Mann et al. International Standards for Terminology and Diagnostic Criteria for Pathological Changes in Rats and Mice (INHAND)[M]. Translated by Yang Lifeng, Zhou Xiangmei, and Zhao Deming. Beijing: China Agriculture Press, 2019.4.5. Statistical analysis

[0341] 4.5.1. PK results The experimental data were described using mean (Mean) and standard deviation (SD) using Microsoft Excel 2010.

[0342] 4.5.2 Analysis of Lp-PLA2 activity in plasma. The experimental data were collected using Biotek Synergy H1, Software Version Gen 5 software, and analyzed and processed using GraphPad Prism 9 and Microsoft Excel 2010. The statistical method used was “Ordinary one-way ANOVA, Dunnett's multiple comparisons test” in GraphPad Prism 9 to analyze the differences between the groups. The statistical results are expressed as mean ± standard error, and p < 0.05 indicates that the statistical results are significantly different. SR13 inhibits Lp-PLA2 activity in plasma of STZ / LPS ALI rats IC 50 The values ​​were analyzed by nonlinear regression using GraphPadPrism 9 (log(inhibitor) vs. response-Variable slope).

[0343] 4.5.3. Albumin levels and leukocyte differential counts in BALF were analyzed using GraphPad Prism 9 and Microsoft Excel 2010 software. The statistical method was to analyze the differences among the groups using "Ordinary one-way ANOVA, Dunnett's multiple comparisons test" in GraphPad Prism 9. The statistical results were expressed as mean ± standard error, and p < 0.05 indicated that the statistical results were significantly different.

[0344] 4.5.4. Pathological analysis of lung tissues. The scoring data of the pathological injury index of each lung tissue were analyzed using Microsoft Excel and GraphPad Prism5. 2way ANOVA Multiple comparisons were used to perform statistical analysis on the control group, model group and drug-treated group. The data were expressed as mean and standard error, and p<0.05 indicated that the difference was statistically significant.

[0345] 5. Experimental results and discussion

[0346] 5.1. PK testing in plasma samples

[0347] 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.

[0348] 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.

[0349] Table 23. SR13 concentration in SD rat blood

[0350] Group N number Mean concentration (ng / mL) Standard Deviation (SD) STZ / LPS+1mg / kg SR13 10 4.49 4.19 STZ / LPS+3mg / kg SR13 10 22.15 23.74 STZ / LPS+10mg / kg SR13 8 122.26 101.18

[0351] 5.2 Results of the determination of Lp-PLA2 activity in plasma

[0352] The results of the test of Lp-PLA2 activity in plasma showed that the activity of Lp-PLA2 in plasma of STZ / LPS ALI rats was significantly increased (p<0.001). The activity of Lp-PLA2 in plasma of rats in the 1, 3, and 10 mg / kg / day SR13 administration groups decreased by 34.40%, 39.47%, and 68.88% respectively compared with the vehicle group (STZ / LPS+Vehicle). SR13 inhibited the activity of Lp-PLA2 in plasma in a dose-dependent manner, as shown in Table 24 and Fig.19 .

[0353] Table 24. SR13 inhibits Lp-PLA2 activity in plasma of STZ / LPS ALI rats

[0354]

[0355] 5.3. SR13 inhibits the infiltration of inflammatory cells and albumin in BALF of STZ / LPS ALI rats

[0356] leak

[0357] In the SR13 time-dependent efficacy evaluation experiment, the total level of leukocytes in BALF was compared with that in The 10mg / kg SR13 group significantly increased (p<0.05). Compared with the STZ / LPS+Vehicle group (LPS-Day 3), the overall level of leukocytes in BALF decreased by 38.27%; 5 days after LPS induction, the total level of leukocytes in BALF decreased significantly compared with 3 days after induction, and the infiltration of inflammatory cells in the lungs decreased. Compared with the STZ / LPS+Vehicle group (LPS-Day5), the 10mg / kg SR13 group decreased by 24.71%, and the infiltration of inflammatory cells in the lungs further decreased, as shown in Tables 25 and Fig. 20 .

[0358] Compared with the level of white blood cells in BALF of the 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.

[0359] Table 25. SR13 inhibits pulmonary leukocyte infiltration

[0360]

[0361] In the SR13 dose-dependent efficacy evaluation experiment, compared with the STZ / LPS+Vehicle group, the monocytes in the BALF of the 1mg / kg, 3mg / kg and 10mg / kg SR13 administration groups decreased by 17.14%, 26.19% and 51.65%, respectively; the lymphocytes in the BALF decreased by 5.84% and 23.83% in the 3mg / kg and 10mg / kg SR13 administration groups, respectively. SR13 inhibited the infiltration of mononuclear macrophages and lymphocytes in the BALF in a dose-dependent manner. At the same time, the BALF neutrophils decreased by 30.52% and 19.83% in the 1mg / kg and 10mg / kg SR13 administration groups, respectively, as shown in Table 26. Fig.21 .

[0362] Table 26. Inhibitory effect of SR13 on pulmonary monocytes / lymphocytes / neutrophils

[0363]

[0364]

[0365] In the SR13 dose-dependent efficacy evaluation experiment, the total albumin level in BALF was also detected 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 Vehicle group 5 days after LPS stimulation. Compared with the Vehicle group, the albumin in BALF of the 1mg / kg, 3mg / kg and 10mg / kg SR13 administration groups decreased by 31.38%, 27.86% and 37.54%, respectively. The results showed that 10mg / kg SR13 significantly inhibited the leakage of albumin in BALF and the destruction of the alveolar-capillary barrier, as shown in Table 27 and Fig. 22 .

[0366] Table 27. SR13 inhibits albumin leakage in BALF

[0367]

[0368]

[0369] 5.4. SR13 improves lung tissue pathological damage in STZ / LPS ALI rats

[0370] According to pathological examination, 3 days after LPS induction, the samples in the control group showed mild alveolar wall thickening, accompanied by a small amount of inflammatory cell infiltration, mainly neutrophils, and mild pulmonary edema in some samples. Compared with the control group, the alveolar wall thickening in the model group was aggravated, the range was enlarged, and the alveolar structure was unclear; the alveolar epithelial cells were significantly damaged, the inflammatory cell infiltration was aggravated, and the inflammatory cells were mainly monocytes, neutrophils and macrophages; the degree of pulmonary edema was aggravated, and the number of samples with pulmonary edema increased; some samples showed mild vascular damage. Compared with the model group, the lesions in the drug-treated group showed slight improvement, but there was no statistical difference in the single lesion index, see Table 25.

[0371] Five days after LPS induction, some samples in the control group showed mild mononuclear cell infiltration and alveolar wall thickening. The model group had obvious lung inflammation, but compared with the model group 3 days after LPS induction, the degree of alveolar wall thickening was reduced and the range was reduced; the degree of inflammatory cell infiltration was reduced, vascular damage disappeared, alveolar epithelial cell damage was reduced, pulmonary edema was slightly aggravated, and the overall injury score was reduced, indicating that the model had a certain natural improvement. Five days after LPS induction, the degree of alveolar wall thickening and alveolar epithelial damage in the drug-treated group was reduced compared with the model group, and the range was reduced; the degree of inflammatory cell infiltration was reduced, and the drug-treated group was reduced compared with the model group in all lesions, but there was no statistical difference in the single lesion index, see Table 28.

[0372] The overall score analysis of lung tissue pathology showed that 5 days after LPS induction, the improvement rate of the drug group was 43.24% compared with the model group, which was 20.94% higher than the natural improvement rate. The statistical analysis results showed that 5 days after LPS induction, the drug could significantly improve the pathological damage of lung tissue (p<0.01) through 2wayANOVA Multiple comparisons statistical analysis, as shown in Table 29 and Fig.23 .

[0373] Table 28. Analysis results of various lung tissue injury indicators

[0374]

[0375] Remarks: Administration group: 10 mg / kg SR13.

[0376] Table 29. Analysis results of overall lung tissue injury score

[0377]

[0378] 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 (%) = (score 模型组-LPS-Day 3 -score 模型组-LPS-Day 5 ) / score 模型组-LPS-Day 3 *100, improvement rate of drug-treated group (%) = (score 模型组-LPS-Day 3 -score 给药组-LPS-Day 5 ) / score 模型组-LPS-Day 3 *100. Note: The administration group is 10 mg / kg SR13.

[0379] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the 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 ingredient also includes ceftazidime; optionally, the pharmaceutical preparation includes a liquid preparation and a solid preparation.

Images