Application of Epiblastin A in preparation of medicine for relieving cisplatin-induced acute kidney injury

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses a novel application of Epiblastatin A in preparation of a medicine for relieving cisplatin-induced acute kidney injury. The application disclosed by the invention for the first time reveals that by inhibiting the activity of Casein Kinase 1 (CK1) kinase, Epiblastine A can obviously relieve renal dysfunction and kidney tissue structure damage caused by cis-platinum. Experiments prove that the compound can effectively reduce serum creatinine and urea nitrogen levels, significantly improve renal tubule pathological injury, down-regulate expression of renal injury early-stage specific biomarkers KIM-1 and NGAL, and definitely inhibit renal tubule epithelial cell apoptosis in an in-vitro model. The invention also provides a specific pharmaceutical composition containing the Epiblastin A and a preparation form of the specific pharmaceutical composition. The invention provides a brand new mechanism and a high-efficiency and low-toxicity treatment strategy for clinically preventing and treating cisplatin dosage-limited renal toxicity, and has important clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of Epiblastin A in the preparation of a drug for alleviating cisplatin-induced acute kidney injury. Background Technology

[0002] In the 1970s, cisplatin began to be widely used clinically to treat various malignant tumors, including bladder cancer, cervical cancer, head and neck malignancies, and small cell or non-small cell lung cancer. It is one of the most effective and commonly used chemotherapy drugs for treating solid tumors. However, its clinical application is greatly limited by the series of serious adverse reactions it causes, such as damage to normal tissues and organs, while simultaneously killing tumor cells. The main adverse reactions of cisplatin include nephrotoxicity and a range of systemic organ damage. Nephrotoxicity is the most common; approximately one-third of patients treated with cisplatin develop renal dysfunction leading to acute renal failure. This dose-related nephrotoxicity significantly limits the clinical application of cisplatin. For a long time, the mechanism by which cisplatin causes damage to normal cells has not been fully understood. Numerous studies have found that DNA damage, inflammatory mediators, necrosis, ferroptosis, apoptosis, oxidative stress, and autophagy may all contribute to the damage to normal tissues and organs caused by cisplatin. [ 1 ] .

[0003] Although platinum-based chemotherapy drugs have been gradually replaced by newer chemotherapy drugs in recent years, traditional platinum-based drugs and their derivatives are still widely used in clinical anti-tumor chemotherapy due to their effectiveness. However, the toxic side effects of platinum-based drugs, such as kidney damage, greatly limit their clinical use. Although some drugs, such as amifostine, are currently used to alleviate kidney damage caused by cisplatin chemotherapy, the use of amifostine itself is limited due to a series of side effects, including vomiting and a tendency to cause hypotension. [ 2 ] Therefore, developing new, effective, and non-toxic drugs that can improve cisplatin-induced acute kidney injury is of great clinical significance.

[0004] Casein Kinase 1 (CK1) is an ATP-competitive serine / threonine protein kinase whose function may be related to apoptosis, cell cycle regulation, and DNA damage repair. [ 3 ] Nevertheless, the role of CK1 in kidney disease, especially acute kidney injury, has not yet been reported.

[0005] References: 1 Ozkok A, Edelstein CL. Pathophysiology of cisplatin-induced acute kidney injury. BioMed research international 2014; 2014: 967826. 2 Duan Z, Cai G, Li J, Chen X. Cisplatin-induced renal toxicity in elderly people. Ther Adv Med Oncol 2020; 12: 1758835920923430. 3 Janovska P, Normant E, Miskin H, Bryja V. Targeting Casein Kinase 1(CK1) in Hematological Cancers. Int J Mol Sci 2020; 21. Summary of the Invention

[0006] This invention provides the use of the CK1 protein kinase inhibitor Epiblastin A in the preparation of drugs for acute kidney injury. The use of this inhibitor in the preparation of drugs to alleviate symptoms related to cisplatin-induced acute kidney injury solves the technical problem that there are no suitable drugs in the prior art for treating cisplatin-induced damage to kidney structure and function.

[0007] More specifically, the present invention aims to provide the use of Epiblastin A in the preparation of a medicament for alleviating symptoms associated with cisplatin-induced acute kidney injury.

[0008] This invention explores the use of EpiblastinA, a serine / threonine protein kinase inhibitor, in alleviating symptoms associated with cisplatin-induced acute kidney injury (AKI) by applying it to cisplatin-induced acute kidney injury (AKI) in mouse and in vitro cell models. Results showed that intervention with EpiblastinA in both cisplatin-induced mouse and in vitro cell models significantly improved renal structure and function, and reduced renal tubular cell apoptosis in AKI patients. Our findings have the potential to provide an effective clinical drug for the prevention and treatment of AKI.

[0009] To achieve the objectives of this invention, the invention includes the following technical solutions: Use of Epiblastin A in the preparation of drugs for alleviating cisplatin-induced acute kidney injury.

[0010] Furthermore, in the above-described uses, Epiblastin A exerts its effects by inhibiting the activity of Casein Kinase 1 (CK1) kinase.

[0011] Furthermore, in the above-described uses, the dosage of the drug is from 5 mg / kg / day to 20 mg / kg / day, preferably 10 mg / kg / day.

[0012] Furthermore, in the above-described uses, the drug is administered via intraperitoneal injection.

[0013] The present invention also discloses a pharmaceutical composition for alleviating cisplatin-induced acute kidney injury, the composition comprising a therapeutically effective amount of Epiblastin A and a pharmaceutically acceptable carrier.

[0014] Furthermore, in the above-described pharmaceutical composition, the pharmaceutically acceptable carrier comprises DMSO, PEG300, TWEEN80, and physiological saline.

[0015] Furthermore, the above-mentioned pharmaceutical composition is formulated as an injection.

[0016] Furthermore, in the above-mentioned pharmaceutical composition, the concentration of Epiblastin A in the composition is from 0.1 mg / mL to 10 mg / mL.

[0017] Furthermore, the above-mentioned pharmaceutical composition includes a pillbox, the pillbox comprising: a) A first component, which is a pharmaceutical composition as described in any of the preceding claims; and b) The second component is cisplatin; c) Optionally, include instructions for use to guide the combined or sequential administration of the pharmaceutical composition with cisplatin. Compared with the prior art, the present invention has the following outstanding advantages: This invention reveals for the first time the crucial role of CK1 kinase in cisplatin-induced acute kidney injury and innovatively develops its inhibitor, Epiblastin A, into a highly effective and low-toxicity renal protective drug. This invention overcomes the bottleneck of existing clinical protective agents with significant side effects and limited efficacy, exhibiting multiple significant beneficial effects: Epiblastin A, by specifically inhibiting CK1 kinase, intervenes in the disease process from a key upstream node, significantly improving cisplatin-induced renal dysfunction and renal tissue structural damage. This compound not only effectively inhibits renal tubular cell apoptosis but also significantly downregulates early-specific biomarkers of acute kidney injury, enabling early intervention and precise treatment of kidney damage. Furthermore, the optimized pharmaceutical composition provided by this invention is stable, has high bioavailability, does not produce significant toxic side effects at effective doses, and exhibits excellent safety, providing a novel solution for the safe clinical application of cisplatin chemotherapy and possessing significant clinical translational value. Attached Figure Description

[0018] Figure 1 : Figure 1 The left figure shows the statistical results of serum urea nitrogen in mice after treatment with solvent, 5 mg / kg / day, 10 mg / kg / day and 20 mg / kg / day EPA in a cisplatin model; the right figure shows the statistical results of serum creatinine in mice. Figure 1 The left image (B) shows representative PAS staining images of kidney glycogen in mice in a model of cisplatin treatment with solvent and 10 mg / kg / day EPA. The right image corresponds to a statistical chart of pathological damage scores based on PAS staining. Figure 2 : Figure 2 A shows the levels of KIM1 and NGAL mRNA in kidney tissues of a mouse model treated with solvent and 10 mg / kg / day EPA by quantitative real-time PCR. Figure 2 The top figure (B) shows the levels of KIM1 and NGAL proteins in kidney tissue of a mouse model treated with solvent and 10 mg / kg / day EPA by western blot. The bottom figure shows the grayscale statistics of the protein bands. Figure 3 : Figure 3 A shows the statistical graph of cell viability as detected by CCK8 assay after 24 h of treatment with different concentrations (0, 2.5, 5, 10, 20, 50 mM) of Epiblastin A in renal tubular epithelial cells in vitro. Figure 3 The left image (B) shows a representative apoptosis assay in renal tubular epithelial cells after treatment with solvent and 5mM EPA combined with stimulation with cisplatin 5mg / ml for 24h in vitro, detected by flow cytometry. The right image shows the statistical results of apoptosis. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Materials and methods 1) Materials and reagents Epiblastin A (catalog number: HY-114858, purity: 99.44%) was purchased from MCE (USA), NGAL antibody from Abcam, and KIM-1 antibody from R&D Systems. qPCR assay kits were purchased from Nanjing Novizan Pharmaceutical Co., Ltd.; apoptosis assay kits were purchased from B&D Pharmaceuticals. Eight-week-old male C57BL / 6J mice were purchased from Nanjing Jicui Pharmaceutical Co., Ltd.; mouse renal tubular epithelial cells were purchased from ATCC (catalog number: CRL-3361).

[0021] 2) Cell culture and treatment Mouse renal tubular epithelial cells (mPTCs) were cultured in DMEM / F12 medium containing 10% fetal bovine serum, 0.5% penicillin, and streptomycin at 37°C, with 5% carbon dioxide and 95% air. To investigate the role of epiblastin A in cisplatin-induced renal tubular epithelial cell damage, when mPTCs reached 70% confluence, the medium was switched to DMEM / F12 without fetal bovine serum, and pretreated with Epiblastin A (dissolved in DMSO, solubility 15.81 mM) for 1 h, or treated with DMSO in the same way. After incubating in an incubator for 24 h, cells were collected for flow cytometry analysis of apoptosis and other experiments.

[0022] 3) Cisplatin-induced acute kidney injury mouse model and experimental grouping Eight-week-old male C57BL / 6J mice were randomly divided into seven groups of six each: a blank control group (vehicle), a 10 mg / kg Epiblastin A (EPA) group, a 20 mg / kg Epiblastin A (EPA) group, a cisplatin injection model group, and groups receiving cisplatin + 5 mg / kg Epiblastin A (EPA), cisplatin + 10 mg / kg Epiblastin A (EPA), and cisplatin + 20 mg / kg Epiblastin A (EPA). Epiblastin A was dissolved in 10% DMSO + 40% PEG300 + 5% TWEEN80 + 45% Saline (volume ratio) and administered via intraperitoneal injection. Mice in the control group or Epiblastin A treatment group were administered either the solvent or Epiblastin A intraperitoneally once daily starting 48 hours before cisplatin injection until sacrifice and tissue collection. Cisplatin model mice received a single intraperitoneal injection of 20 mg / kg cisplatin, and were sacrificed and collected 72 hours after cisplatin injection. Mice in the control group received the same dose of saline. After 72 hours, all mice were euthanized, and kidney and blood samples were collected. All animal experiments complied with the Chinese Regulations on the Management and Use of Laboratory Animals and were approved ethically by the Animal Ethics Committee of Nanjing Medical University (Approval No. IACUC:2310025-1).

[0023] 4) Kidney function test After centrifugation, serum components were collected from mouse blood samples, and serum creatinine and blood urea nitrogen levels were measured using a fully automated biochemical analyzer at Nanjing Children's Hospital.

[0024] 5) Renal tubular injury score The degree of tubular damage was observed in the PAS pathological staining of the kidney, and the tubular damage was assessed according to the semi-quantitative damage scoring criteria. Normal renal tubular tissue was scored as 0 points, renal tubular damage less than 30% was scored as 1 point, renal tubular damage between 30% and 60% was scored as 2 points, and renal tubular damage greater than 60% was scored as 3 points.

[0025] 6) Histological and immunofluorescence staining Kidney tissue was fixed with paraformaldehyde, wrapped in paraffin, and then sectioned for histological staining.

[0026] 7) Western blot of proteins Proteins were extracted from kidney tissue following literature procedures. Western blot results were analyzed using ImageJ software for grayscale analysis.

[0027] 8) Flow cytometry detection of apoptosis Mouse renal tubular epithelial cells were cultured, pretreated with Epiblastin A for 1 h, and then treated with cisplatin for 24 h. The apoptosis level of the cells was analyzed by flow cytometry. The cell experiments were repeated three times and the results were statistically analyzed.

[0028] 9) Statistical Analysis Data are expressed as mean ± SD. One-way ANOVA was used for comparisons among multiple groups, and the t-test was used for comparisons between two groups. A p-value < 0.05 was considered statistically significant.

[0029] Example 1

[0030] Epiblastin A improves kidney injury and renal function in a cisplatin-induced acute kidney injury model.

[0031] To evaluate the role of Epiblastin A in protecting against cisplatin-induced acute kidney injury, we treated mice with different doses of Epiblastin A (5, 10, and 20 mg / kg / day) to induce a kidney model. We then measured renal-related biochemical indicators in the mouse serum. 72 hours after intraperitoneal injection of cisplatin, serum creatinine (Scr: 102.3±14.9 μM in the Vehicle+cis group) and blood urea nitrogen (BUN: 66.4±16.7 mM in the Vehicle+cis group) were significantly elevated, and renal pathological damage, such as tubular dilation and necrosis, was also more severe. However, after treatment with 10 and 20 mg / kg Epiblastin A, the corresponding renal damage and renal function indicators decreased significantly (serum creatinine 10 mg / kg EPA+cis: 56.3±20.4 μM, 20 mg / kg EPA+cis: 54.7±18.8 μM; blood urea nitrogen 10 mg / kg EPA+cis: [missing value]). 42.6±11.7 mM, 20 mg / kg EPA+cis: 44.2±12.5 mM). Although 5 mg / kg of Epiblastin A also had some protective effect, it was not statistically significant. 20 mg / kg did not provide better tubule protection than 10 mg / kg of Epiblastin A. Therefore, we chose 10 mg / kg / day as the subsequent Epiblastin A dosage. Figure 1(A, 1B). Furthermore, the renal tubular injury score also indicated that Epiblastin A treatment could improve cisplatin-induced renal pathological damage. Therefore, Epiblastin A can not only improve renal function but also alleviate renal pathological damage. These results suggest that Epiblastin A can protect against cisplatin-induced acute kidney injury, and it itself has no significant toxic side effects.

[0032] To further demonstrate the role of Epiblastin A in protecting against cisplatin-induced acute kidney injury, we examined early-stage specific biomarkers for acute kidney injury, NGAL and KIM-1. Figure 2 QRT-PCR analysis showed that KIM-1 and NGAL were significantly upregulated in the kidneys of mice with cisplatin-induced acute kidney injury, and their expression levels were significantly decreased after treatment with 10 mg / kg Epiblastin A. Western blot results ( Figure 2 B) This further validated the above results, indicating that Epiblastin A can improve cisplatin-induced acute kidney injury.

[0033] Example 2

[0034] In an in vitro model of renal tubular epithelial cells treated with cisplatin, Epiblastin A treatment improved cisplatin-induced apoptosis.

[0035] In in vitro cultured renal tubular epithelial cells, different concentrations (0, 2.5, 5, 10, 20, 50 μM) of Epiblastin A were added for 24 h. CCK8 assays showed that Epiblastin A did not cause cytotoxicity at concentrations below 50 mM. Figure 3 A); Furthermore, using in vitro cultured renal tubular epithelial cells, pretreatment with Epiblastin A (5 μM) for 1 h was followed by treatment with 5 μg / ml cisplatin for another 24 h. Cells were then harvested and apoptosis levels were detected by flow cytometry. The results showed that Epiblastin A significantly improved cisplatin-induced apoptosis ( Figure 3 B).

[0036] In summary, this invention provides the use of the CK1 inhibitor Epiblastin A in the preparation of a drug for alleviating cisplatin-induced acute kidney injury-related symptoms. The drug is administered via intraperitoneal injection at a mouse dose of 10 mg / kg / day. By inhibiting CK1 kinase activity, it improves apoptosis and thus alleviates acute kidney injury.

[0037] Summary of Examples: This invention quantitatively verified the significant protective effect of Epiblastin A through in vitro and in vivo experiments. In a mouse model of cisplatin-induced acute kidney injury, compared with the model group, treatment with 10 mg / kg Epiblastin A significantly reduced serum creatinine (Scr) and blood urea nitrogen (BUN) levels by approximately 45% and 36%, respectively (Scr from 102.3 μM to 56.3 μM; BUN from 66.4 mM to 42.6 mM), and the renal tissue pathological damage score decreased by 48% (from 2.5 to 1.3). Simultaneously, the mRNA expression levels of early damage markers KIM-1 and NGAL in renal tissue were inhibited by 60%-65%, and their protein expression levels decreased by more than 55%. In an in vitro cell model, 5 μM of Epiblastin A significantly reduced cisplatin-induced renal tubular epithelial cell apoptosis. Dosage experiments showed that 5-20 mg / kg was the effective dose range, and no compound self-toxicity was observed within this range. The above data fully demonstrate the clear efficacy and superiority of this invention.

[0038] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. Use of Epiblastin A in the preparation of drugs for alleviating cisplatin-induced acute kidney injury.

2. The use according to claim 1, characterized in that, Epiblastin A exerts its effect by inhibiting the activity of CaseinKinase 1 (CK1) kinase.

3. The use according to claim 1, characterized in that, The dosage of the drug is from 5 mg / kg / day to 20 mg / kg / day, preferably 10 mg / kg / day.

4. The use according to claim 1, characterized in that, The drug is administered via intraperitoneal injection.

5. A pharmaceutical composition for alleviating cisplatin-induced acute kidney injury, characterized in that, The composition comprises a therapeutically effective amount of Epiblastin A and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, characterized in that, Pharmaceutically acceptable carriers include DMSO, PEG300, TWEEN80, and physiological saline.

7. The pharmaceutical composition according to claim 6, characterized in that, The volume ratio of DMSO, PEG300, TWEEN80 and physiological saline is (5-15):(30-50):(1-10):(30-60).

8. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition is formulated as an injection.

9. The pharmaceutical composition according to claim 5, characterized in that, The concentration of Epiblastin A in the composition is from 0.1 mg / mL to 10 mg / mL.

10. A medicine box, characterized in that, The medicine box includes: a) A first component, which is the pharmaceutical composition according to any one of claims 5 to 9; and b) The second component is cisplatin; c) Optionally, include instructions for use to guide the combined or sequential administration of the pharmaceutical composition with cisplatin.

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