Application of thiazole carboxamide derivative in preparation of medicine for treating acute lung injury / acute respiratory distress syndrome

Abstract

The invention discloses application of a known compound, namely a thiazole carboxamide derivative (with the CAS number of 933886-42-1), in preparation of a medicine for treating acute lung injury / acute respiratory distress syndrome, the compound is an SLC39A1 agonist, zinc ions entering cells can be effectively increased, and by reducing the expression quantity of lung tissue inflammation genes IL-1beta and TNF-alpha of mice, the lung tissue inflammation genes IL-1beta and TNF-alpha can be effectively inhibited, so that the acute lung injury / acute respiratory distress syndrome can be effectively inhibited, and the acute lung injury / acute respiratory distress syndrome can be effectively inhibited. Therefore, lung cell infiltration in ALI / ARDS mice is reduced, oxidative damage and tissue damage of lung tissue, inflammatory factor expression and cell connection are improved, and a new choice is provided for treatment of ALI / ARDS.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug discovery, and particularly relates to application of a thiazole carboxamide derivative in preparation of a drug for treating acute lung injury / acute respiratory distress syndrome. BACKGROUND

[0002] Acute respiratory distress syndrome (ARDS) is a clinical syndrome characterized by refractory hypoxemia after direct or indirect acute lung injury (ALI). From the severe acute respiratory syndrome (SARS) in 2003 to the Middle East respiratory syndrome (MERS) in 2012, and to the novel coronavirus infection (COVID-19) in the winter of 2019, the incidence rates of ARDS in the three outbreaks were 20%, 20-30% and 18-30%, respectively. Many ventilation strategies for ARDS have been proposed, such as low tidal volume, higher positive end-expiratory pressure (PEEP), prone position, neuromuscular blockade and extracorporeal membrane oxygenation. These supportive treatments of protective lung ventilation aim to give patients the opportunity to repair lung damage. Although the development of treatment strategies such as protective mechanical ventilation technology has improved the mortality rate of ARDS patients, there is still no effective drug to reduce the mortality rate. Pathophysiological studies suggest that inflammation, coagulation, oxidative stress and epithelial damage are key changes in the progression of ALI / ARDS, but there is no breakthrough progress, and there is still no effective ALI / ARDS treatment drug for clinical use. Therefore, it is of great significance to analyze the pathogenesis of ALI / ARDS, find new therapeutic targets and develop new targeted compounds for the treatment of ALI / ARDS. SUMMARY

[0003] The present application aims at the problem that there is no specific drug for acute lung injury / acute respiratory distress syndrome and only supportive treatment such as mechanical ventilation, and provides an effect principle of a G748-0185 targeting SLC39A1 agonist that can significantly improve lung inflammatory cell infiltration, protect lung epithelial cell tight junction and improve lung tissue damage by increasing intracellular zinc ion level to regulate inflammatory pathways. The present application provides a theoretical basis and scientific basis for the clinical application of treating acute lung injury / acute respiratory distress syndrome by reducing the expression mechanism of inflammation-related pathway molecules.

[0004] To achieve the above-mentioned purpose, the technical solutions of the present application are as follows: The use of a thiazolium carboxamide derivative in the preparation of a medicament for treating acute lung injury / acute respiratory distress syndrome, wherein the thiazolium carboxamide derivative has the CAS number 933886-42-1; the present invention is named G748-0185, and its chemical name is 5-[(2-Chlorobenzoyl)amino]-2-[(4-methylphenyl) [amino]-4-thiazolecarboxamide; the corresponding Chinese translation is 5-[(2-chlorobenzoyl)amino]-2-[(4-methylphenyl)amino]-4-thiazolecarboxamide; the structural formula is shown in I. .

[0005] Furthermore, the acute lung injury or acute respiratory distress syndrome includes acute lung injury and acute respiratory distress syndrome induced by bacteria, lipopolysaccharide, or cecal ligation and puncture.

[0006] Furthermore, the thiazocarboxamide derivatives exert their effects by stimulating the zinc transporter SLC39A1, thereby increasing the intracellular zinc ion concentration.

[0007] Furthermore, the target organisms of the drug include mammals.

[0008] Furthermore, the mammals include humans, rats, monkeys, rabbits, cats, dogs, sheep, horses, cattle, and pigs.

[0009] Furthermore, the drug can be administered via tracheal infusion, injection, or inhalation; specifically, for mice, the administration method is tracheal drip.

[0010] Furthermore, the dosage form of the drug is any one of an inhaler, a nebulizer, or a solution.

[0011] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0012] Furthermore, the excipients include one or more of the following: carrier, osmotic pressure regulator, suspending agent, solubilizer, pH adjuster, preservative, etc.

[0013] The beneficial effects of this invention include at least the following: The present application shows that SLC39A1 agonist G748-0185 can increase intracellular zinc ion levels, alleviate LPS / IL-1β-induced cell injury in vitro and LPS or CLP-induced ALI / ARDS mice in vivo. The present application found that tight junction proteins were significantly damaged in LPS / IL-1β-induced cell injury in vitro, and the addition of G748-0185 can protect tight junction proteins; in LPS or CLP-induced ALI / ARDS mice in vivo, the increase in inflammatory damage in lung tissue, and intratracheal instillation of G748-0185 can reduce LPS or CLP-induced inflammatory cell infiltration, lung tissue pathological damage, lung tissue inflammatory factor expression and cell junction protection. This beneficial effect may be achieved by acting on the reduction of immune cell infiltration and inhibition of inflammatory response. In summary, the present application suggests the potential of SLC39A1 agonist G748-0185 in the intervention of ALI / ARDS, and provides new possibilities for the development of more effective and safer drugs for the treatment of ALI / ARDS.

[0014] The present application provides a new molecule G748-0185 for relieving ALI / ARDS disease, which can reduce lung cell infiltration in ALI / ARDS mice, improve lung tissue oxidative damage and tissue damage, inflammatory factor expression and cell junction. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Figure for detecting whether G748-0185 can activate SLC39A1 to promote intracellular zinc ion transport using zinc ion fluorescent indicator FluoZin-3, AM.

[0016] Figure 2 Figure for the effect of G748-0185 on cell tight junction ZO-1 protein in A549 cell damage model.

[0017] Figure 3 Figure for the effect of G748-0185 on cell tight junction Occludin protein in A549 cell damage model.

[0018] Figure 4 Figure for the effect of G748-0185 on lung inflammatory cell infiltration in LPS and CLP-induced ALI / ARDS mice. A and C are cell counts in the lung alveolar lavage fluid of LPS and CLP-induced ALI / ARDS mice, respectively, and B and D are protein contents in the lung alveolar lavage fluid.

[0019] Figure 5Representative pictures of hematoxylin staining of lung tissue of G748-0185 treated LPS induced ALI / ARDS mice. Top panel, 20x, scale bar = 500 μm; middle panel, 100x, scale bar = 100 μm; bottom panel, 400x, scale bar = 20 μm.

[0020] Figure 6 Representative pictures of hematoxylin staining of lung tissue of G748-0185 treated CLP induced ALI / ARDS mice. Top panel, 20x, scale bar = 500 μm; middle panel, 100x, scale bar = 100 μm; bottom panel, 400x, scale bar = 20 μm.

[0021] Figure 7 Effect of G748-0185 on lung tissue inflammatory gene expression of LPS and CLP induced ALI / ARDS mice. The mRNA levels of the determined inflammatory genes are: A and C are the expression levels of lung tissue inflammatory gene interleukin-1β (IL-1β) of LPS and CLP induced ALI / ARDS mice, respectively, B and D are the expression levels of lung tissue inflammatory gene tumor necrosis factor-α (TNF-α) of LPS and CLP induced ALI / ARDS mice, respectively. Data are expressed as mean ± standard deviation, n = 5, *P < 0.05; **P < 0.01; ***P < 0.001.

[0022] Figure 8 Effect of G748-0185 on lung tissue epithelial cell tight junction of LPS and CLP induced ALI / ARDS mice. A and B are the immunofluorescence of ZO-1 and Occludin protein in lung tissue epithelial cells of LPS induced ALI / ARDS mice, C and D are the immunofluorescence of ZO-1 and Occludin protein in lung tissue epithelial cells of CLP induced ALI / ARDS mice 16 h, E and F are the immunofluorescence of ZO-1 and Occludin protein in lung tissue epithelial cells of CLP induced ALI / ARDS mice 48 h. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0024] In addition, the technical solutions among various embodiments of the present application can be combined with each other, but it must be based on the fact that a person skilled in the art can realize the combination, and when the combination of technical solutions appears contradictory or unachievable, it should be considered that the combination of technical solutions does not exist and is not within the protection scope required by the present application.

[0025] The solutions proposed by the present application are specifically described below through specific embodiments: Embodiment 1 The material selection and experimental method are specifically as follows: (1) Experimental cells Select A549 cell strain, and culture in DMEM containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator.

[0026] (2) Experimental animals Select 80 C57BL / 6J male mice of 8 weeks old, and keep the room temperature at 23±1°C and the humidity at 50±60%. The light and dark cycle is 12 hours, and the mice can obtain sterile special mouse feed and water at will.

[0027] (3) Experimental method G748-0185 (CAS No.: 933886-42-1, chemical name: 5-[(2-Chlorobenzoyl)amino]-2-[(4-methylphenyl)amino]-4-thiazolecarboxamide) activated A549 cell SLC39A1 model: A549 cells were added with different concentrations of G748-0185 (0, 1 μM, 4 μM, 7 μM, 10 μM, purchased from ChemDiv company, No.: G748-0185, CAS No.: 933886-42-1), and the intracellular zinc ion fluorescence intensity was detected after 12 h of treatment.

[0028] LPS / IL-1β induced A549 cell injury model: A549 cells were divided into Blank, LPS / IL-1β, LPS / IL-1β+G748-0185 and LPS / IL-1β+G748-0185+TPEN four groups, and LPS / IL-1β (LPS 1 μg / mL, IL-1β 1 ng / mL) was added at 0 h for treatment, and then G748-0185 (10 μM) and zinc ion chelator TPEN (1 μM) were immediately added. After 12 h of LPS / IL-1β induction, the cells were collected for immunofluorescence staining.

[0029] LPS intratracheal instillation mouse model: C57BL / 6J mice were divided into PBS, DMSO, LPS, LPS+G748-0185 and LPS+G748-0185+TPEN intratracheal instillation groups, 8 mice in each group. 1 hour before modeling, 200 μL (10 mg / Kg) TPEN was injected intraperitoneally. On the first day, 50 μL (1 mg / Kg) LPS was intratracheally instilled in each group, and then 50 μL (1 mg / Kg) G748-0185 was immediately intratracheally instilled. The control group was instilled with the same volume of PBS or DMSO. 72 hours after LPS intratracheal instillation, lung tissue and bronchoalveolar lavage fluid were taken for H&E staining, immunofluorescence staining, QPCR and other tests.

[0030] CLP mouse model: C57BL / 6J mice were divided into Sham, CLP, CLP+DMSO, CLP+G748-0185 and CLP+G748-0185+TPEN groups, 8 mice in each group. 1 hour before modeling, 200 μL (10 mg / Kg) TPEN was injected intraperitoneally. On the first day, CLP modeling was performed in each group (after anesthesia, the skin tissue and peritoneum on the midline of the abdomen were incised, the cecum was found in the abdominal cavity, and a small amount of feces was squeezed out at 1 / 2 of the cecum. After the operation, the cecum was returned and the abdominal cavity was closed. The Sham group performed the same operation but without ligation and puncture. Immediately after CLP, 50 μL (1 mg / Kg) LPS was intratracheally instilled, and then 50 μL (1 mg / Kg) G748-0185 was immediately intratracheally instilled. The control group was instilled with the same volume of DMSO. 16 hours after modeling, lung tissue and bronchoalveolar lavage fluid were taken for H&E staining, immunofluorescence staining, QPCR and other tests. To further evaluate the effect of modeling and drug on disease progression, lung tissue was collected 48 hours after modeling for H&E staining, immunofluorescence staining and apoptosis-related tests.

[0031] The A549 cell zinc ion fluorescence intensity parameter evaluation is as follows: Discard the old culture medium of A549 cells, wash with PBS for 3 times, and incubate with zinc ion fluorescence indicator FluoZin-3, AM (1 μM) at room temperature for 40 min in the dark. Discard the culture medium, wash with PBS for 3 times, and then detect the intracellular zinc ion fluorescence intensity.

[0032] The A549 cell tight junction protein analysis is as follows: The collected A549 cell climbing sheets were fixed with 4% paraformaldehyde for 10 min, washed with PBS at the minimum speed of the shaker for 3 times, 5 min each time, after washing, 5% goat serum was used for room temperature blocking for 1 h, after blocking, the primary antibody (1:200 dilution) was incubated at 4°C overnight, after incubation of the primary antibody, PBST was used for washing 3 times, 5 min each time, after washing, fluorescent secondary antibody (1:500 dilution) and nuclear dye DAPI (1:200 dilution) were used for room temperature incubation for 2 h, after incubation, PBST was used for washing 3 times, 5 min each time, and then anti-fluorescence quenching agent was used for mounting and fixing. Observation was carried out under a fluorescence microscope at 600 times magnification.

[0033] The lung tissue inflammation and cell infiltration parameters were evaluated as follows: The bronchoalveolar lavage samples were centrifuged at 2000 r / min twice, and cell counts were immediately detected, and the supernatant was subjected to protein concentration (BCA) analysis.

[0034] The histopathological analysis was as follows: The collected lung tissue samples were fixed with 4% paraformaldehyde.

[0035] Hematoxylin and eosin staining (H&E): The lung tissue paraffin sections were baked in an oven at 65°C for at least 1 hour to ensure that the paraffin on the tissue sections was fully melted. Then, the sections were placed in xylene twice to remove the melted paraffin and were hydrated in gradient ethanol. The sections were stained with hematoxylin and eosin, respectively, and then were placed in an oven at 65°C to dry the excess water on the sections, and then were fixed with neutral resin. The stained sections were observed under an optical microscope at 100 times and 400 times magnification, respectively.

[0036] The mRNA analysis of inflammatory factors in lung tissue was as follows: The expression of inflammatory factors in lung tissue was determined using QPCR: interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α).

[0037] The lung tissue epithelial cell tight junction protein analysis was as follows: The frozen sections were warmed at room temperature for 30 min, washed in PBS for 3 times, 5 min each time, and placed in sodium citrate antigen repair solution for antigen repair in a microwave oven for 8 min at medium heat and 7 min at low heat, cooled at room temperature for 20-30 min, washed in PBS for 3 times, 5 min each time, 10% goat serum was added on each sample for blocking at room temperature for 1 h, the primary antibody (1:200 dilution) was used for incubation at 4°C overnight, washed with PBST for 3 times, 5 min each time, and then the fluorescent secondary antibody (1:500 dilution) and the nuclear dye DAPI (1:200 dilution) were used for incubation at room temperature in the dark for 2 h, washed with PBST for 3 times, 5 min each time, and then mounted and fixed with an anti-fluorescence quencher. Observation was performed under a fluorescence microscope at 600 times magnification.

[0038] Statistical analysis was performed as follows: Significant differences between groups were compared by one-way analysis of variance (ANOVA), and data were expressed as mean ± standard deviation (S.D.). All statistical tests were performed using Prism 8 software (GraphPad Software, San Diego, CA, USA). P<0.05 was considered significant, and N.S. indicated no significant difference.

[0039] The following are the results of experimental data analysis: Figure 1 To detect whether G748-0185 can activate SLC39A1 to promote intracellular zinc ion transport using the zinc ion fluorescent indicator FluoZin-3, AM. As shown in the figure, the fluorescence intensity of intracellular zinc ions increases with the increase of the concentration gradient of compound G748-0185, indicating that G748-0185 can activate SLC39A1 to promote the transport of intracellular zinc ions.

[0040] Figure 2 and Figure 3 The effect of G748-0185 on cell tight junction in the A549 cell damage model. Figure 2 and Figure 3 The immunofluorescence of ZO-1 and Occludin proteins, respectively, as shown in the figure, compared with the control group Blank, after induction by adding LPS / IL-1β, the two tight junction proteins were significantly reduced, after further adding G748-0185 treatment, the tight junction proteins were significantly increased, and finally after adding TPEN, this effect was obviously reversed.

[0041] Figure 4Effect of G748-0185 on lung inflammatory cell infiltration in LPS and CLP induced ALI / ARDS mice. A and C are the cell counts in the bronchoalveolar lavage fluid of LPS and CLP induced ALI / ARDS mice, respectively, compared with the control group LPS and CLP group, the cell infiltration in the bronchoalveolar lavage fluid of LPS+G748-0185 and CLP+G748-0185 groups is reduced, and this effect is obviously reversed after further adding TPEN; B and D are the protein content of the mouse bronchoalveolar lavage fluid, the total protein content in the bronchoalveolar lavage fluid of LPS+G748-0185 and CLP+G748-0185 groups is reduced, and this effect is obviously reversed after further adding TPEN; the data is expressed as mean ± standard deviation, n = 5, *P < 0.05; **P < 0.01; ***P < 0.001.

[0042] Figure 5 and 6 Effect of G748-0185 on lung tissue damage in LPS and CLP induced ALI / ARDS mice. The figure is a representative picture of hematoxylin and eosin (H&E) staining of mouse lung tissue. It can be seen from the figure that LPS and CLP obviously cause lung tissue inflammatory cell infiltration, alveolar septum thickening and alveolar collapse, compared with the pathological damage of the lung in the LPS and CLP group, the pathological damage of the lung in the LPS+G748-0185 and CLP+G748-0185 groups is alleviated, and this effect is obviously reversed after further adding TPEN. Therefore, the supplementation of SLC39A1 agonist G748-0185 can alleviate the pathological damage of mouse lung tissue caused by LPS or CLP.

[0043] Figure 7 Effect of G748-0185 on lung tissue inflammatory gene expression in LPS and CLP induced ALI / ARDS mice. Figure 7 A and 7C are the determination of the mRNA level of lung tissue inflammatory gene interleukin-1β (IL-1β) in LPS and CLP induced ALI / ARDS mice, respectively, Figure 7 B and 7D are the determination of the mRNA level of lung tissue inflammatory gene tumor necrosis factor-α (TNF-α) in LPS and CLP induced ALI / ARDS mice, respectively. It can be seen from the figure that the mRNA expression level of IL-1β, TNF-α in the lung tissue of LPS+G748-0185 and CLP+G748-0185 groups of mice is significantly lower than that of LPS or CLP treated mice; this effect is obviously reversed after further adding TPEN. Therefore, SLC39A1 agonist G748-0185 can significantly alleviate the lung tissue inflammation of mice caused by LPS or CLP.

[0044] Figure 8The effect of G748-0185 on the tight junction of lung tissue epithelial cells of ALI / ARDS mice induced by LPS and CLP at different times. It can be seen from the figure that the ZO-1 and Occludin in the lung tissue of mice in the LPS+G748-0185 and CLP+G748-0185 groups are obviously more than that in the LPS or CLP treated mice; after further adding TPEN, this effect is obviously reversed. Therefore, the SLC39A1 agonist G748-0185 can effectively protect the destruction of the tight junction protein of the lung tissue epithelial cells of mice caused by LPS or CLP.

[0045] Result analysis: Acute lung injury (ALI) / acute respiratory distress syndrome (ARDS) is a common critical illness of the respiratory system caused by multiple factors, which has a serious adverse effect on patients, families and society, and has attracted widespread attention in the medical field. The current clinical treatment of ALI / ARDS mainly relies on supportive treatment, such as mechanical ventilation, prone position and corticosteroids. Seeking more effective treatment methods is still the goal of scholars. SLC39A1 is a key membrane protein responsible for regulating zinc ion transfer into the cell, which is highly expressed in type II alveolar epithelial cells. Its agonist G748-0185 can significantly increase the intracellular zinc ion concentration and reduce the lung alveolar lavage cell infiltration, pathological tissue damage, lung tissue inflammatory factor expression and protect the cell tight junction induced by LPS and CLP. According to the above experimental results, it is speculated that the anti-SLC39A1 agonist G748-0185 can alleviate ALI / ARDS by reducing lung inflammation.

[0046] In summary, the present application shows that the SLC39A1 agonist G748-0185 can increase the intracellular zinc ion level and has an alleviating effect on LPS / IL-1β induced cell injury in vitro and LPS or CLP induced ALI / ARDS mice in vivo. The present application found that the tight junction protein was significantly damaged in LPS / IL-1β induced cell injury in vitro, and the addition of G748-0185 can protect the tight junction protein; in LPS or CLP induced ALI / ARDS mice in vivo, the lung tissue inflammation damage increased, and the tracheal instillation of G748-0185 can reduce the inflammatory cell infiltration, lung tissue pathological damage, lung tissue inflammatory factor expression and cell connection caused by LPS or CLP. This beneficial effect may be achieved by reducing immune cell infiltration and inhibiting inflammatory response. In general, the present application suggests the potential of SLC39A1 agonist G748-0185 in the intervention of ALI / ARDS, and provides a new possibility for developing more effective and safer drugs for treating ALI / ARDS.

[0047] It should be noted that, in this document, the terms "comprising", "comprises" or any other variation thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can also include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element preceded by "comprises a" does not, without more constraints, foreclose the existence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[0048] The above-mentioned embodiment numbers of the present application are only for description, not representing the advantages and disadvantages of the embodiments.

[0049] The above embodiments are only used to illustrate the technical solutions of the present application and not limit the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced equivalently without departing from the spirit and scope of the present application, and all of them should be covered in the scope of the claims of the present application.

Claims

1. Use of a thiazole carboxamide derivative with CAS No. 933886-42-1 in the manufacture of a medicament for treating acute lung injury / acute respiratory distress syndrome.

2. Use according to claim 1, characterized in that, The acute lung injury or acute respiratory distress syndrome includes acute lung injury and acute respiratory distress syndrome induced by bacteria, lipopolysaccharide or cecal ligation puncture.

3. Use according to claim 1, characterized in that, The thiazole carboxamide derivative exerts its effect by agonizing zinc transporter SLC39A1 to increase intracellular zinc ion concentration.

4. Use according to claim 1, characterized in that, The subject of the medicament includes mammals.

5. Use according to claim 4, characterized in that, The mammals include human, mouse, monkey, rabbit, cat, dog, sheep, horse, cow, pig.

6. Use according to claim 1, characterized in that, The administration mode of the medicament includes intratracheal administration, injection administration and inhalation administration.

7. The use according to claim 1, characterized in that, The dosage form of the medicament is any one of inhalant, atomizer or solution.

8. Use according to claim 7, characterized in that, The medicament further includes pharmaceutically acceptable adjuvants.

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