AKI therapeutic agent and application of MRS2578 in preparation of therapeutic agent

Abstract

The invention relates to application of MRS2578 in preparation of an acute kidney injury therapeutic agent, and further relates to the acute kidney injury therapeutic agent containing the MRS2578, and a medicament group containing the acute kidney injury therapeutic agent and used for treating solid tumors. Cis-platinum is a chemotherapeutic agent which is very commonly used in tumor treatment, but complication acute kidney injury caused by application of the cis-platinum seriously damages health of patients. The invention proves that the MRS2578 can be used for treating and relieving acute kidney injury caused by cisplatin application, and a potential new drug is provided for relieving complication in chemotherapy. According to the present invention, it is expected that MRS2578 and cis-platinum can be combined in chemotherapy so as to alleviate or even eliminate acute kidney injury in the tumor killing process.

Description

Technical Field

[0001] The present invention relates to the field of treatment of tumors and their complications, and more particularly, to the use of MRS2578 in the preparation of an acute kidney injury therapeutic agent, and an acute kidney injury therapeutic agent containing MRS2578. Background Art

[0002] Cisplatin is a commonly used broad-spectrum anticancer drug in clinical practice, which has inhibitory effects on various solid tumors such as lung cancer, nasopharyngeal carcinoma, and ovarian cancer. The renal transport of cisplatin is regulated by proximal tubule transporters, and its accumulation in renal proximal tubule epithelial cells leads to cell damage and even death. Therefore, many patients receiving cisplatin chemotherapy will show complications of acute kidney injury (AKI).

[0003] P2Y6 (pyrimidinergic receptor P2Y6, encoded by the p2ry6 gene) is one of the important members of the purinergic receptor family. It can mediate changes in related pathways by recognizing extracellular UDP and has attracted much attention in the fields of neurodegenerative diseases, vasculitis, autoimmune inflammation, etc. There are many aspects of research on P2Y6. P2Y6 deficiency can inhibit vascular inflammation and atherosclerosis in mice. P2Y6 promotes angiotensin II-induced hypertension in mice, and knocking out P2Y6 can significantly reduce blood pressure, oxidative stress and endothelial cell dysfunction. In the angiotensin II-induced abdominal aortic aneurysm (AAA) model, P2Y6 inhibitors instead promote inflammatory responses and MMP expression, thereby aggravating AAA progression and tumor rupture. P2Y6 deficiency can also improve glucose tolerance and insulin sensitivity, reduce systemic inflammation, and thus prevent obesity in mice. P2Y6 deficiency promotes NK cell and T cell activation, and aggravates experimental autoimmune encephalomyelitis and DSS-induced IBD by enhancing TH17 cell activation. In DSS-induced IBD, P2Y6 inhibitors show the effect of inhibiting enteritis.

[0004] The above studies show that the research on the function of P2Y6 in inflammation is still limited and controversial, and P2Y6 may play completely opposite effects in different inflammatory events. There is no literature report on the function and molecular mechanism of P2Y6 in the kidney.

[0005] MRS2578(C 20 H 20 N6S4, N,N"-1,4-butanediyl-bis[N'-(3-isothiocyanatophenyl)thiourea), has a molecular formula as shown in Formula I and is a highly selective P2Y6 receptor antagonist. Currently, there is no research on its use in the treatment of AKI.

[0006] Summary of the invention

[0007] Based on the clinical problem of cisplatin-induced AKI and the potential role of P2Y6 receptors in inflammation and cell damage, the present invention proposes a new strategy for treating acute kidney injury using the P2Y6 receptor antagonist MRS2578. Through subsequent experimental studies, the present invention confirms that MRS2578 has a significant alleviating and therapeutic effect on cisplatin-induced AKI, providing a potential new drug for alleviating this complication in chemotherapy.

[0008] In a specific embodiment, the acute kidney injury is cisplatin-induced acute kidney injury.

[0009] In a specific embodiment, the P2Y6 receptor antagonist is MRS2578.

[0010] The present invention also provides an acute kidney injury therapeutic agent, comprising a P2Y6 receptor antagonist.

[0011] In a specific embodiment, the acute kidney injury is cisplatin-induced acute kidney injury.

[0012] In a specific embodiment, the P2Y6 receptor antagonist is MRS2578.

[0013] The present invention also provides a pharmaceutical composition for treating solid tumors, comprising cisplatin and an acute kidney injury therapeutic agent, wherein the acute kidney injury therapeutic agent is a P2Y6 receptor antagonist.

[0014] In a specific embodiment, the P2Y6 receptor antagonist is MRS2578.

[0015] In a specific embodiment, the dose of MRS2578 is no less than 3 mg / kg.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. Provides a new strategy for the treatment of acute kidney injury (AKI):

[0018] This invention reveals for the first time the important role of P2Y6 receptor in AKI and confirms that the P2Y6 receptor antagonist MRS2578 has significant alleviating and therapeutic effects on cisplatin-induced AKI. This discovery provides new drug targets and strategies for the treatment of AKI, which is expected to improve the treatment effect of clinical AKI patients.

[0019] 2. Broadened the application scope of P2Y6 receptor antagonists:

[0020] As an important member of the purinergic receptor family, P2Y6 receptor has been studied in the fields of neurodegenerative diseases, vasculitis, autoimmune inflammation, etc., but there is still a gap in the research on kidney diseases. The present invention broadens the application scope of P2Y6 receptor antagonists by confirming the effectiveness of MRS2578 in the treatment of AKI, and provides clues for its potential application in other kidney diseases.

[0021] 3. Provides potential new drugs for complications of AKI during chemotherapy:

[0022] Cisplatin is a commonly used broad-spectrum anticancer drug in clinical practice, but the AKI complication caused by it seriously affects the health and quality of life of patients. The present invention confirms that MRS2578 can effectively alleviate cisplatin-induced AKI, providing a potential new drug for reducing or eliminating this complication in chemotherapy, and is expected to improve the tolerance and efficacy of chemotherapy.

[0023] 4. Provides new ideas for drug combination:

[0024] The present invention also proposes a drug group for treating solid tumors, including cisplatin and an acute kidney injury therapeutic agent (P2Y6 receptor antagonist). The idea of ​​this drug combination not only takes into account the treatment of tumors, but also takes into account the complications that may occur during chemotherapy, providing a more comprehensive consideration for clinical medication.

[0025] 5. Sufficient experimental evidence and potential for clinical application:

[0026] The present invention fully confirms the therapeutic effect of MRS2578 on cisplatin-induced AKI through in vitro and in vivo experiments, including the detection of biochemical function level, tissue structure level and molecular marker level. These experimental evidences provide strong support for the clinical application of MRS2578 and have broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a clinical correlation study of P2Y6 expression in renal tissue of AKI patients. GSE1536 is a publicly published Microarray data of renal biopsy tissue of renal transplant patients. Patients were divided into two groups according to whether renal dysfunction occurred after surgery: a good renal function group and an acute renal dysfunction group (including patients with and without rejection). Figure A shows the gene expression level of P2Y6 in renal tissue of the two groups of patients, B shows the correlation analysis between the expression level of P2Y6 in patients and glomerular filtration rate, and C shows the correlation analysis between the expression level of P2Y6 in patients and serum creatinine level.

[0028] Figure 2This is a study on the expression changes of P2Y6 in AKI mouse models and cell models, where A is the publicly published renal tissue RNAseq data of various AKI mouse models, and the figure compares the mRNA expression of P2Y6 in the renal tissue of mice in the three model groups and the control group; B is the expression and localization of P2Y6 after cisplatin stimulation of renal tubular epithelial cells, and C is the expression of P2Y6 protein level and mRNA level in renal tubular epithelial cells under cisplatin stimulation;

[0029] Figure 3 This is a study on apoptosis of renal tubular epithelial cells after stimulation with cisplatin alone or with cisplatin and P2Y6 agonist UDP. A is flow cytometry to detect apoptosis, and B is a statistical graph.

[0030] Figure 4 This is a study on cellular oxidative stress and mitochondrial function after overexpression of P2Y6 in renal tubular epithelial cells, where A is the level of reactive oxygen species and B is the change in mitochondrial membrane potential;

[0031] Figure 5 This is a study on how MRS2578 alleviates cisplatin-induced AKI in mice, where A is the injection picture and the molecular formula of MRS2578, B is the administration situation, and C and D are statistical graphs of serum BUN and Scr levels, respectively;

[0032] Figure 6 PAS staining of mouse renal tubules (A) and detection of NGAL protein levels (B);

[0033] Figure 7 TUNEL staining of rat renal tubules shows apoptosis and pyroptosis of tubular cells;

[0034] Figure 8 This study investigated the effect of low-dose MRS2578 on alleviating cisplatin-induced AKI in mice. DETAILED DESCRIPTION

[0035] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0036] Example 1

[0037] Study on the role of P2Y6 receptor in AKI.

[0038] 1. P2Y6 is upregulated in the renal tissue of patients with renal dysfunction.

[0039] The human kidney disease genotype / phenotype database Nephroseq was used to retrieve more clinical AKI data, and DNA microarray data of renal tissues of 22 renal transplant patients were obtained (GSE1563). By analyzing the correlation between P2Y6 gene expression and clinical practice, it was found that P2Y6 expression in renal biopsy tissues of patients with acute renal dysfunction and acute rejection was significantly higher than that in renal tissues with normal function, and was negatively correlated with glomerular filtration rate and positively correlated with patients' serum creatinine levels (e.g. Figure 1 shown).

[0040] 2. P2Y6 expression was significantly upregulated in the renal tissue of the AKI mouse model and the acute tubular epithelial cell injury model.

[0041] We used the renal tissue transcriptomic data of three AKI mouse models in the GEO database (GSE153625) to analyze and found that the mRNA expression of P2Y6 in the renal tissue of the three AKI mice was significantly upregulated. Next, by culturing the renal tubular epithelial cell line in vitro and stimulating it with cisplatin, we found that the expression of P2Y6 in renal tubular epithelial cells was significantly upregulated after cisplatin stimulation (e.g. Figure 2 shown).

[0042] 3. Activation of P2Y6 aggravates cisplatin-induced cell apoptosis.

[0043] Renal tubular epithelial cell lines were cultured in vitro and treated with cisplatin for 2 hours before being treated with the P2Y6 receptor stimulator UDP. Cells were harvested 24 hours later and flow cytometry was used to detect cell apoptosis. The results showed that activation of P2Y6 significantly aggravated cisplatin-induced cell apoptosis (e.g. Figure 3 shown).

[0044] 4. Overexpression of P2Y6 induces oxidative stress and mitochondrial damage in renal tubular epithelial cells.

[0045] Renal tubular epithelial cell lines were cultured in vitro, transfected with P2Y6 expression vectors, and the cellular ROS content and mitochondrial membrane potential were detected by fluorescent probes. The results showed that overexpression of P2Y6 significantly increased the ROS level and significantly decreased the mitochondrial membrane potential (e.g. Figure 4 shown).

[0046] Based on the above experimental results, we can draw the following conclusions: P2Y6 receptor is upregulated in acute kidney injury (AKI) and is associated with the severity of renal dysfunction; activation of P2Y6 receptor aggravates apoptosis of renal tubular epithelial cells, indicating that activation of P2Y6 receptor has a harmful effect on renal tubular epithelial cells; overexpression of P2Y6 leads to oxidative stress and mitochondrial damage, which further supports the negative effect of P2Y6 receptor in AKI; therefore, both activation and overexpression of P2Y6 receptor are associated with the negative effects of AKI, and therefore inhibiting the activity of P2Y6 receptor is likely to help alleviate or treat AKI. In Example 2, we selected P2Y6 receptor antagonist MRS2578 for further verification.

[0047] Example 2

[0048] Study on the role of P2Y6 receptor antagonist MRS2578 in AKI.

[0049] 1. Effects of MRS2578 on AKI mice

[0050] Eight-week-old wild-type male mice C57BL / 6 were randomly divided into three groups. The mice were pre-medicated two days in advance. The control group and the model group were given an equal amount of MRS2578 drug solvent, and the intervention group was given MRS2578 (30 mg / kg / day). Two days later, the model group and the intervention group were intraperitoneally injected with cisplatin (25 mg / kg) to prepare the AKI model, and the control group was intraperitoneally injected with an equal amount of normal saline. The intervention group was intraperitoneally injected with MRS2578 again 24 and 48 hours after modeling, and the control group and the model group were given an equal amount of solvent. The mice were euthanized 72 hours after the cisplatin injection and the samples were collected ( Figure 5 A and B), venous blood and renal tissue samples were collected. Blood biochemistry was performed to detect BUN and Scr levels. The results are shown in Figure 5 As shown in C and D, injection of MRS2578 could significantly reduce the increase in BUN and Scr levels caused by cisplatin administration.

[0051] PAS observed the pathological damage of renal tubules. Figure 6 As shown in middle A, the degree of tubular pathological damage was significantly reduced.

[0052] Western blotting was used to detect the expression of renal tubular injury factor NGAL. Figure 6 As shown in B, the expression of NGAL in the model group increased significantly, while that in the drug intervention group was inhibited.

[0053] The apoptosis of renal cells was detected by TUNEL staining. Figure 7As shown in the figure, the number of apoptotic cells in the model group increased significantly, while the injection of MRS2578 could significantly reduce the number of apoptotic cells in the renal tubules; at the same time, the level of pyroptosis-related protein N-GSDMD also decreased significantly.

[0054] 2. Effect of low-dose MRS2578 on AKI

[0055] Eight-week-old wild male C57BL / 6 mice were selected, and the dosage was reduced. The cisplatin-induced AKI mouse model was constructed by the method of administering drugs at the same time as modeling. The mice were randomly divided into four groups. The control group and the model group were given an equal amount of MRS2578 drug solvent. The two intervention groups were intraperitoneally injected with 3 mg / kg MRS2578 and 10 mg / kg MRS2578, respectively. One hour later, the two intervention groups and the model group were intraperitoneally injected with cisplatin (25 mg / kg) to construct the AKI model, and the control group was given an equal amount of normal saline. The intervention group was intraperitoneally injected with MRS2578 again 24 and 48 hours after modeling. Euthanasia was performed 72 hours after cisplatin injection to obtain venous blood and kidney specimens. Scr and BUN were detected, and PAS staining of renal tissue was performed to detect tubular pathological damage.

[0056] The results are as follows Figure 8 As shown, short-term pretreatment with low-dose MRS2578 can also alleviate cisplatin-induced renal dysfunction and tubular damage.

[0057] The above experimental results show that the injection of 3-30 mg / kg of MRS2578 into mice can alleviate cisplatin-induced renal injury, whether at the biochemical function level, tissue structure level or molecular marker level. Therefore, it can be considered that MRS2578 has a therapeutic effect on cisplatin-induced renal injury. In addition, since the above experiments show that the combined administration of MRS2578 and cisplatin can alleviate and treat cisplatin-induced acute renal injury, this also means that the two can be administered in combination to treat corresponding tumors, so the present invention should also cover such a combined combination of agents.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of P2Y6 receptor antagonists in the preparation of therapeutic agents for acute kidney injury.

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

3. The use according to claim 1, characterized in that: The P2Y6 receptor antagonist is MRS2578.

4. An acute kidney injury therapeutic agent, characterized in that Contains a P2Y6 receptor antagonist.

5. The acute kidney injury therapeutic agent according to claim 4, characterized in that The acute kidney injury is cisplatin-induced acute kidney injury.

6. The therapeutic agent for acute kidney injury according to claim 4, characterized in that The P2Y6 receptor antagonist is MRS2578.

7. A pharmaceutical composition for treating solid tumors, characterized in that: The invention comprises cisplatin and an acute kidney injury therapeutic agent, wherein the acute kidney injury therapeutic agent is a P2Y6 receptor antagonist.

8. The pharmaceutical composition according to claim 7, characterized in that: The P2Y6 receptor antagonist is MRS2578.

9. The pharmaceutical composition according to claim 8, characterized in that: The dose of MRS2578 is no less than 3 mg / kg.

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