Application of thioamide compound in preparation of medicine for treating acute kidney injury

Abstract

The invention relates to an application of a compound 2-(2-(5-methyl-2-oxotetrahydrofuran-3-yl) acetyl) hydrazine-1-thioformamide in preparation of a medicine for treating acute kidney injury, in particular to an application of the compound 2-(2-(5-methyl-2-oxotetrahydrofuran-3-yl) acetyl) hydrazine-1-thioformamide. The compound provided by the invention can effectively reduce cell injury and inflammatory response, and reduce expression and secretion levels of cell inflammatory factors; the traditional Chinese medicine composition can effectively improve the pathological level of kidney tissues of acute kidney injury animals in vivo, inhibit protein expression of a kidney injury factor KIM-1, reduce the levels of creatinine and urea nitrogen in serum of AKI mice and inhibit recruitment of inflammatory cells such as macrophages and neutrophils, so that kidney inflammations are relieved, and the traditional Chinese medicine composition has excellent prevention and treatment effects on acute kidney injury and inflammations.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceuticals, and particularly relates to the use of a thioamide compound in the preparation of a medicament for treating acute kidney injury. Background Art

[0002] Acute kidney injury (AKI) is a clinical syndrome characterized by a rapid decline in renal function within hours. Approximately 100 million people worldwide suffer from AKI every year, and its incidence is still rising. Among inpatients, the incidence of AKI is 7.2%-11.6%, and 28.6% of patients experience a second recurrence of AKI. Severe AKI patients have a high mortality rate and poor prognosis, and are an important cause of secondary chronic kidney disease, renal failure and death of inpatients, bringing a serious economic and social burden.

[0003] During the process of AKI, ischemia, the use of nephrotoxic drugs, sepsis, urinary tract obstruction, etc. can all cause irreversible loss of renal tubular epithelial cells and the entire nephron. The main pathological features are persistent inflammation, excessive oxidative stress and programmed death of renal tubular epithelial cells. At present, there is an urgent lack of specific drugs for preventing and treating acute kidney injury clinically. Therefore, it is of great significance to search for kidney-protective drugs that can reduce tissue damage and promote the repair of tubular cells. Summary of the Invention

[0004] To solve the above technical problems, the present invention includes the following aspects:

[0005] The first aspect of the present invention provides the use of a compound in the preparation of a medicament for preventing or treating acute kidney injury, and the compound is 2-(2-(5-methyl-2-oxotetrahydrofuran-3-yl)acetyl)hydrazine-1-carbothioamide.

[0006] Preferably, the structural formula of the compound is as follows: (Compound Cpd-155).

[0007] Preferably, the acute kidney injury is cisplatin-induced acute kidney injury or ischemia-reperfusion-induced acute kidney injury.

[0008] The second aspect of the present invention provides the use of a pharmaceutical composition in the preparation of a medicament for preventing or treating acute kidney injury, and the pharmaceutical composition contains the compound 2-(2-(5-methyl-2-oxotetrahydrofuran-3-yl)acetyl)hydrazine-1-carbothioamide and pharmaceutically acceptable excipients.

[0009] Preferably, the dosage form of the pharmaceutical composition is tablets, capsules, granules, injections, patches, gels or ointments. More preferably, the dosage form of the pharmaceutical composition is tablets or capsules.

[0010] Preferably, the concentration of the compound in the pharmaceutical composition is 20 - 160 μM. More preferably, the concentration of the compound in the pharmaceutical composition is 20 - 80 μM. Even more preferably, the concentration of the compound in the pharmaceutical composition is 40 - 80 μM.

[0011] Preferably, the acute kidney injury is cisplatin-induced acute kidney injury or ischemia-reperfusion-induced acute kidney injury.

[0012] The third aspect of the present invention provides an application of a compound in the preparation of a drug for preventing or treating inflammatory diseases, and the compound is 2-(2-(5-methyl-2-oxotetrahydrofuran-3-yl)acetyl)hydrazine-1-carbothioamide.

[0013] Preferably, the structural formula of the compound is as follows: (Cpd-155 compound).

[0014] The fourth aspect of the present invention provides an application of a pharmaceutical composition in the preparation of a drug for preventing or treating inflammatory diseases, and the pharmaceutical composition contains the compound 2-(2-(5-methyl-2-oxotetrahydrofuran-3-yl)acetyl)hydrazine-1-carbothioamide and a pharmaceutically acceptable excipient.

[0015] Preferably, the dosage form of the pharmaceutical composition is tablets, capsules, granules, injections, patches, gels or ointments. More preferably, the dosage form of the pharmaceutical composition is tablets or capsules.

[0016] Preferably, the concentration of the compound in the pharmaceutical composition is 20 - 160 μM. More preferably, the concentration of the compound in the pharmaceutical composition is 20 - 80 μM. Even more preferably, the concentration of the compound in the pharmaceutical composition is 40 - 80 μM.

[0017] The fifth aspect of the present invention provides an application of a compound in the preparation of a drug for reducing the levels of inflammatory factor TNF-α, chemokine CCL2 and / or chemokine CXCL1 in serum, and the compound is 2-(2-(5-methyl-2-oxotetrahydrofuran-3-yl)acetyl)hydrazine-1-carbothioamide.

[0018] Preferably, the structural formula of the compound is as follows: (Cpd-155 compound).

[0019] Preferably, the present invention provides an application of the compound in the preparation of a drug for simultaneously reducing the levels of inflammatory factor TNF-α, chemokine CCL2 and chemokine CXCL1 in serum.

[0020] The sixth aspect of the present invention provides an application of a pharmaceutical composition in the preparation of a drug for reducing the levels of inflammatory factor TNF-α, chemokine CCL2 and / or chemokine CXCL1 in serum, wherein the pharmaceutical composition comprises the compound 2-(2-(5-methyl-2-oxotetrahydrofuran-3-yl)acetyl)hydrazine-1-carbothioamide and pharmaceutically acceptable excipients.

[0021] Preferably, the dosage form of the pharmaceutical composition is tablet, capsule, granule, injection, patch, gel or ointment. More preferably, the dosage form of the pharmaceutical composition is tablet or capsule.

[0022] Preferably, the concentration of the compound in the pharmaceutical composition is 20-160 μM. More preferably, the concentration of the compound in the pharmaceutical composition is 20-80 μM. Further preferably, the concentration of the compound in the pharmaceutical composition is 40-80 μM.

[0023] Preferably, the present invention provides an application of the pharmaceutical composition in the preparation of a drug for simultaneously reducing the levels of inflammatory factor TNF-α, chemokine CCL2 and chemokine CXCL1 in serum.

[0024] Technical effects produced by the present invention:

[0025] The present invention unexpectedly finds that the compound 2-(2-(5-methyl-2-oxotetrahydrofuran-3-yl)acetyl)hydrazine-1-carbothioamide (Cpd-155) can effectively reduce cell damage and inflammatory response, reduce the expression and secretion levels of cell inflammatory factors; and effectively improve the pathological level of kidney tissues in animals with acute kidney injury in vivo, inhibit the protein expression of kidney injury factor KIM-1, reduce the levels of creatinine and blood urea nitrogen in the serum of AKI mice, and inhibit the recruitment of inflammatory cells such as macrophages and neutrophils, thereby reducing kidney inflammation, and having excellent preventive and therapeutic effects on acute kidney injury and inflammation. Description of the drawings

[0026] Figure 1 is the nuclear magnetic resonance spectrum of Cpd-155;

[0027] Figure 2 is the mass spectrum of Cpd-155;

[0028] Figure 3 is the comparison chart of the effects of different concentrations of Cpd-155 on the viability of HK2 cells;

[0029] Figure 4 is the comparison chart of the effects of different concentrations of Cpd-155 on the injury of renal tubular epithelial HK2 cells induced by cisplatin in vitro experiments;

[0030] Figure 5Results of Western Blot and Real-time PCR showing that Cpd-155 alleviates hypoxia-reoxygenation-induced injury of renal tubular epithelial cells in vitro;

[0031] Figure 6 Results showing that Cpd-155 inhibits the levels of inflammatory factors induced by H / R in vitro;

[0032] Figure 7 Results showing that prophylactic administration of Cpd-155 reduces the levels of serum creatinine and blood urea nitrogen in mice with acute kidney injury induced by ischemia-reperfusion;

[0033] Figure 8 Results showing that prophylactic administration of Cpd-155 reduces the pathological injury of the kidneys in mice with acute kidney injury induced by ischemia-reperfusion;

[0034] Figure 9 Results showing that prophylactic administration of Cpd-155 reduces the levels of inflammatory factors in the kidney tissues of mice with acute kidney injury model;

[0035] Figure 10 Results of staining the heart, liver, spleen and lung tissues of mice in the simple drug addition group with hematoxylin and eosin (H&E) staining kit; Detailed implementation manners

[0036] The present invention will be further described in detail below in combination with experimental examples and the accompanying drawings, but the implementation manners of the present invention are not limited thereto. Unless otherwise specified, the definitions of the significance symbols of P values in the experimental examples and the accompanying drawings of this application are as follows: *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01, P < 0.001.

[0037] Compound 2-(2-(5-methyl-2-oxotetrahydrofuran-3-yl)acetyl)hydrazine-1-carbothioamide used in the experimental examples of the present invention (structural formula is ) was purchased from Shanghai TargetMol Biotechnology Co., Ltd., with the product number AL-281 / 41668155, which is abbreviated as "Cpd-155" in the experimental examples of the specification, and its structural confirmation spectrum is as Figure 1-2 shown.

[0038] Experimental Example 1. Effects of different concentrations of Cpd-155 on the viability of human renal tubular epithelial cells (HK2 cells) in vitro

[0039] HK2 cells in the logarithmic growth phase were seeded into 96-well plates. A normal control group and groups with different concentration gradients of Cpd-155 were set up, and they were cultured with HK2 cells for another 24 - 48 h. The CCK8 method was used to detect the cell proliferation status to judge cell viability. The HK2 cell-specific medium without Cpd-155 was used as the normal control group (NC). The concentrations of Cpd-155 were 1.25, 2.5, 5, 10, 20, 40, 80, 160, and 320 μM. The detection results are as Figure 3 shown. It can be seen from the figure that when the concentration of Cpd-155 was 1.25 - 160 μM, Cpd-155 showed no obvious toxicity to HK2 cells.

[0040] An in vitro cell injury model stimulated by cisplatin was constructed with HK2 cells. Under the treatment with different concentrations of Cpd-155, cisplatin was added and the cells were stimulated for another 24 h. The CCK8 method was used to detect the cell proliferation status to judge cell viability. The HK2 cell-specific medium without Cpd-155 + Cisplatin was used as the normal control group (NC), and the HK2 cell-specific medium without Cpd-155 but containing Cisplatin was used as the injury control (denoted as CIS). The concentration of Cisplatin was 20 μM, and the concentrations of Cpd-155 were 1.25, 2.5, 5, 10, 20, 40, 80, 160 μM. The detection results are as Figure 4 shown. From Figure 4 the CCK8 results, it can be seen that cisplatin caused damage to renal tubular epithelial cells, while Cpd-155 at 20 - 160 μM could significantly reduce the growth inhibitory effect of cisplatin on HK2 cells.

[0041] Experimental Example 2: Protective effect of Cpd-155 on hypoxia-reoxygenation (H / R)-induced renal tubular epithelial cell injury

[0042] HK2 cells in the logarithmic growth phase were seeded into 6-well plates and divided into a normal group (NC), a model group (H / R), a low-dose Cpd-155 group (H / R + Cpd-155 20 μM), a medium-dose Cpd-155 group (H / R + Cpd-155 40 μM), and a high-dose Cpd-155 group (H / R + Cpd-155 80 μM). Each group was repeated 3 - 4 times. The seeding density was approximately 1.0×10 5Cells / ml. After incubation with low, medium, and high doses of Cpd-155 for 12 h, the cells were subjected to hypoxia-reoxygenation treatment. They were cultured under hypoxic conditions (5% CO2, 1% O2, and 94% N2) for 12 h, and then reoxygenated for 6 h (5% CO2 and 95% air). The cells were washed three times with PBS, collected, total protein was extracted, and the protein expression of kidney injury molecule KIM1 was detected by Western Blot and semi-quantitative analysis was performed.

[0043] HK2 cells were seeded in 12-well plates at an inoculation density of approximately 0.5×10 5 cells / well and divided into a normal group (NC), a model group (H / R), a low-dose Cpd-155 group (H / R + Cpd-155 20 μM), a medium-dose Cpd-155 group (H / R + Cpd-155 40 μM), and a high-dose Cpd-155 group (H / R + Cpd-155 80 μM). Each group was repeated 3 - 4 times. After incubation with low, medium, and high doses of Cpd-155 for 12 h, the cells were subjected to hypoxia-reoxygenation treatment. They were cultured under hypoxic conditions (5% CO2, 1% O2, and 94% N2) for 12 h, and then reoxygenated for 6 h (5% CO2 and 95% air). The cells were washed three times with PBS, collected, RNA was extracted, reverse transcribed into cDNA, and amplified.

[0044] The test results of Western Blot and Real-time PCR are shown in Figure 5 Figures A and 5B respectively. The test results showed that after treatment with Cpd-155, the protein level and mRNA level of kidney injury molecule KIM-1 in H / R-stimulated HK2 cells were significantly inhibited, demonstrating that Cpd-155 has a protective effect on cisplatin-induced renal tubular epithelial injury.

[0045] Test Example 3. Effect of Cpd-155 on in vitro inflammatory response of renal tubular epithelial cells induced by hypoxia-reoxygenation (H / R)

[0046] Referring to the method of Test Example 2, the expression levels of inflammatory factor TNF-α and chemokines CCL2 and CXCL1 in HK2 cells treated in each test group were detected by Real time PCR. The test results are shown in Figure 6 Figure. The test results showed that hypoxia-reoxygenation significantly upregulated the mRNA levels of TNF-α, CCL2, and CXCL1 in renal tubular epithelial cells, while the treatment groups with different doses of Cpd-155 significantly inhibited the production of TNF-α, CCL2, and CXCL1, confirming that Cpd-155 can significantly inhibit the inflammatory response induced by hypoxia-reoxygenation.

[0047] Experimental Example 4, Effect of Preventive Administration of Cpd-155 on Serum Creatinine and Blood Urea Nitrogen (BUN) in an Acute Kidney Injury Model of Ischemic Reperfusion Mice

[0048] C57BL / 6 mice at 6-8 weeks of age were adaptively cultured for 1-2 days and divided into a normal control group (NC), an ischemic reperfusion group (I / R), a low-dose group of Cpd-155 (I / R + Cpd-155 12.5 mg / kg), a medium-dose group (I / R + Cpd-155 25 mg / kg), and a high-dose group (I / R + Cpd-155 50 mg / kg), with 6 mice in each group. Cpd-155 was intraperitoneally injected 1 day in advance, and then an ischemic reperfusion mouse model was established. After 24 hours, the mice were sacrificed under anesthesia, and serum samples and kidney tissues were collected. The contents of creatinine and blood urea nitrogen in the serum of the animal model were detected according to the instructions of the creatinine and blood urea nitrogen kits (purchased from Nanjing Jiancheng Bioengineering Institute), and tissue proteins were extracted for Western Blot detection.

[0049] (1) The method for creatinine determination is as follows:

[0050]

[0051] Creatinine content (μmol / L) = [(Measured A2 - K * Measured A1) - (Blank A2 - K * Blank A1)] / [(Standard A2 - K * Standard A1) - (Blank A2 - K * Blank)] * Standard concentration (442 μmol / L)

[0052] Note: Dilution factor K = (Sample volume + Volume of Enzyme Solution A) / (Sample volume + Volume of Enzyme Solution A + Volume of Enzyme Solution B) = 186 / 246

[0053] (2) The method for blood urea nitrogen test is as follows:

[0054]

[0055] Blood urea nitrogen content (mmol / L) = (Measured OD value - Blank measured value) / (Standard OD value - Blank OD value) * Standard concentration (10 mmol / L) * Dilution factor before sample testing

[0056] The test results are as Figure 7 shown. In the ischemic reperfusion-induced model group, the contents of serum creatinine and blood urea nitrogen increased significantly, and renal function deteriorated. However, different doses of Cpd-155 effectively reduced the levels of blood urea nitrogen and serum creatinine in the model group ( Figure 7 Figures 7A and 7B). The results of Western Blot and semi-quantitative analysis showed that after treatment with different concentrations of Cpd-155 in the cisplatin-induced model group, the protein expression level of the kidney injury factor KIM-1 was significantly inhibited ( Figure 7C), indicating that Cpd-155 has good protective effects on acute kidney injury and a low effective dose.

[0057] Experimental Example 5. Effects of Cpd-155 on renal tissue pathology in ischemic reperfusion mice

[0058] 6-8-week-old C57BL / 6 mice were adaptively cultured for 1-2 days and divided into a normal control group (NC), an ischemic reperfusion group (I / R), a low-dose Cpd-155 group (I / R + Cpd-155 12.5 mg / kg), a medium-dose group (I / R + Cpd-155 25 mg / kg), and a high-dose group (I / R + Cpd-155 50 mg / kg), with 6 mice in each group. Cpd-155 was intraperitoneally injected 1 day in advance, and then an ischemic reperfusion mouse model was established. After 24 hours, the mice were sacrificed under anesthesia, and serum samples and kidney tissues were collected.

[0059] The kidney tissues of mice were stained using a glycogen (PAS) staining kit to observe the kidney injury in ischemic reperfusion mice. The staining results are as Figure 8 shown. The experimental results showed that the level of renal tubular injury in the I / R group mice increased significantly, mainly manifested as the dilation of renal tubular lumen, the disappearance of the brush borders of a large number of renal tubular epithelial cells, swelling, shedding, and a large amount of glycogen deposition in the lumen. However, only a small number of epithelial cell integrity was damaged in the Cpd-155 treatment group mice, and the glycogen deposition was significantly reduced, indicating that Cpd-155 treatment had a significant improvement effect on the renal tissue pathology in ischemic reperfusion mice.

[0060] Experimental Example 6. Inhibitory effects of prophylactic administration of Cpd-155 on inflammation in ischemic reperfusion mice

[0061] 6-8-week-old C57BL / 6 mice were adaptively cultured for 1-2 days and divided into a normal control group (NC), an ischemic reperfusion group (I / R), a low-dose Cpd-155 group (I / R + Cpd-71 12.5 mg / kg), a medium-dose group (I / R + Cpd-155 25 mg / kg), and a high-dose group (I / R + Cpd-155 50 mg / kg), with 6 mice in each group. Cpd-155 was intraperitoneally injected 1 day in advance, and then an ischemic reperfusion mouse model was established. After 24 hours, the mice were sacrificed under anesthesia, and serum samples and kidney tissues were collected.

[0062] The ELISA test results are as Figure 9 shown. The experimental results showed that the levels of inflammatory factor TNF-α and chemokines CCL2 and CXCL1 in the serum of ischemic reperfusion mice increased significantly, while Cpd-155 could significantly reduce the release of TNF-α, CCL2, and CXCL1 in the serum of ischemic reperfusion mice, alleviating the inflammatory response.

[0063] Experimental Example 7: Effects of Cpd-155 on the Histopathology of the Heart, Liver, Spleen, Lung, and Kidney Tissues of Normal Mice

[0064] Six normal control mice were intraperitoneally injected with Cpd-155 (80 mg / kg). After 24 hours, the mice were sacrificed under anesthesia, and serum samples and heart, liver, spleen, lung, and kidney tissues were collected. The heart, liver, spleen, and lung tissues of the mice were stained using a hematoxylin and eosin (H&E) staining kit, and the staining results are shown as Figure 10 follows. The test results showed that no tissue damage occurred in the mice in the simple drug administration group, indicating that Cpd-155 had no toxicity to the tissues of normal mice at this dose.

[0065] In summary, as demonstrated by the above experiments, Cpd-155 has good protective effects on kidney injury and inflammatory responses in vivo and in vitro. Therefore, this drug is expected to become a key drug for the prevention and treatment of acute kidney injury.

[0066] Although specific embodiments of the present invention have been described, those skilled in the art should recognize that various changes and modifications can be made to the present invention without departing from the scope or spirit of the present invention. Therefore, the present invention is intended to cover all such changes and modifications that fall within the scope of the appended claims and their equivalents.

Claims

1. Use of a compound in the preparation of a drug for preventing or treating acute kidney injury, characterized in that: The compound is 2-(2-(5-methyl-2-oxotetrahydrofuran-3-yl)acetyl)hydrazine-1-carbothioamide.

2. The use according to claim 1, characterized in that: The acute kidney injury is cisplatin-induced acute kidney injury or ischemia-reperfusion-induced acute kidney injury.

3. Use of a pharmaceutical composition in the preparation of a drug for preventing or treating acute kidney injury, characterized in that: The pharmaceutical composition comprises the compound 2-(2-(5-methyl-2-oxotetrahydrofuran-3-yl)acetyl)hydrazine-1-thiocarboamide and pharmaceutically acceptable excipients.

4. The use according to claim 3, characterized in that: The dosage form of the pharmaceutical composition is tablets, capsules, granules, injections, patches, gels or ointments; preferably, the dosage form of the pharmaceutical composition is tablets or capsules.

5. The use according to claim 3, characterized in that: The concentration of the compound in the pharmaceutical composition is 20-160 μM.

6. The use according to claim 5, characterized in that: The concentration of the compound in the pharmaceutical composition is 20-80 μM.

7. Use of a compound in the preparation of a drug for preventing or treating inflammatory diseases, characterized in that: The compound is 2-(2-(5-methyl-2-oxotetrahydrofuran-3-yl)acetyl)hydrazine-1-carbothioamide.

8. Use of a pharmaceutical composition in the preparation of a drug for preventing or treating an inflammatory disease, characterized in that: The pharmaceutical composition comprises the compound 2-(2-(5-methyl-2-oxotetrahydrofuran-3-yl)acetyl)hydrazine-1-thiocarboamide and pharmaceutically acceptable excipients.

9. Use of a compound in the preparation of a drug for reducing the levels of inflammatory factor TNF-α, chemokine CCL2 and / or chemokine CXCL1 in serum, characterized in that: The compound is 2-(2-(5-methyl-2-oxotetrahydrofuran-3-yl)acetyl)hydrazine-1-carbothioamide.

10. Use of a pharmaceutical composition in the preparation of a drug for reducing the levels of inflammatory factor TNF-α, chemokine CCL2 and / or chemokine CXCL1 in serum, characterized in that: The pharmaceutical composition comprises the compound 2-(2-(5-methyl-2-oxotetrahydrofuran-3-yl)acetyl)hydrazine-1-thiocarboamide and pharmaceutically acceptable excipients.

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