Use of phenoxazine-1-carboxylic acid in the treatment of cisplatin-mediated acute kidney injury

Abstract

The application belongs to the field of clinical drug toxic side effects, and particularly relates to the use of phenazine-1-carboxylic acid in treating cisplatin-mediated acute kidney injury. The application first discovers and proves that phenazine-1-carboxylic acid can effectively alleviate cisplatin-induced acute kidney injury. In a cisplatin-induced acute kidney injury model of mice, intraperitoneal injection of phenazine-1-carboxylic acid can effectively protect kidney function, which is specifically manifested as follows: significantly reducing urea nitrogen and creatinine levels in serum of model mice; reducing pathological injury of kidney tissue and reducing tubular injury score; and inhibiting expression of inflammatory factors and injury markers in kidney tissue. The treatment effect is equivalent to that of a classic ferroptosis inhibitor, Ferrostatin-1, and a new candidate drug for solving the dose-limiting toxicity of cisplatin in clinical application is provided.

Description

Technical Field

[0001] This invention belongs to the field of clinical drug toxicity and side effects, specifically relating to the use of phenazine-1-carboxylic acid in the treatment of cisplatin-mediated acute kidney injury. Background Technology

[0002] Cisplatin is a broad-spectrum chemotherapy drug widely used to treat various solid tumors, such as lung cancer, ovarian cancer, and head and neck cancer. However, cisplatin is accompanied by serious dose-limiting toxicities in clinical use, among which acute kidney injury (AKI) is the most common and prominent. Since cisplatin is mainly excreted through the kidneys, it accumulates at high concentrations in renal tubular epithelial cells, directly causing damage and death of these cells, leading to a sharp decline in renal function, manifested as elevated serum creatinine and blood urea nitrogen levels. This severe nephrotoxicity not only limits the dosage and duration of cisplatin treatment, affecting anti-tumor efficacy, but may also force treatment interruption and even develop into chronic kidney disease, threatening patients' quality of life and prognosis.

[0003] Ferroprelation is a novel form of cell death driven by iron-dependent lipid peroxidation. Numerous studies have investigated its role in cisplatin-induced acute kidney injury (AKI). Pretreatment of mice with the ferroprelation inhibitor ferrostatin-1 significantly reduced serum urea nitrogen and creatinine levels, indicating that inhibiting ferroprelation can alleviate cisplatin-induced kidney damage. However, due to the metabolic instability and poor pharmacokinetics of ferrostatin-1, it has not yet entered drug development. Therefore, there is an urgent need to find small molecules that effectively inhibit ferroprelation to address cisplatin-induced kidney damage.

[0004] However, known ferroptosis inhibitors such as ferstatin-1 generally suffer from poor metabolic stability and undesirable pharmacokinetic properties (e.g., low oral bioavailability), which greatly limits their potential for clinical translation. Therefore, discovering novel ferroptosis inhibitors with good drug-like potential is of urgent clinical need and significant application value for developing drugs to prevent and treat cisplatin-induced AKI. Summary of the Invention

[0005] Despite years of research into the mechanisms of cisplatin nephrotoxicity, highly effective and specific preventative measures remain lacking in clinical practice. Traditional interventions such as hydration, diuresis, and electrolyte supplementation, while effective to some extent, cannot completely prevent kidney damage. Therefore, elucidating its molecular mechanisms and identifying new intervention targets is crucial. Cisplatin inhibits the cysteine / glutamate antitransporter (System Xc-) on the renal tubular epithelial cell membrane, leading to the depletion of intracellular glutathione (GSH), which in turn weakens the activity of glutathione peroxidase 4 (GPX4), ultimately causing the accumulation of lipid peroxides and ferroptosis. Addressing the common problems of poor metabolic stability and unfavorable pharmacokinetic properties in existing ferroptosis inhibitors, this invention aims to screen and identify novel ferroptosis inhibitors with good drug-like potential from a small molecule natural product library.

[0006] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0007] The first aspect of this invention discloses the use of phenazine-1-carboxylic acid in the preparation of a medicament for the prevention and / or treatment of cisplatin-induced acute kidney injury.

[0008] A second aspect of the present invention discloses the use of phenazine-1-carboxylic acid in the preparation of medicaments for the prevention and / or treatment of non-tumor diseases associated with ferroptosis.

[0009] Preferably, the non-tumor-related diseases associated with ferroptosis include ischemia / reperfusion injury, neurodegenerative diseases, drug-induced liver injury, or drug-induced kidney injury.

[0010] A third aspect of the present invention discloses a pharmaceutical composition comprising a therapeutically effective amount of phenazine-1-carboxylic acid and a pharmaceutically acceptable carrier, said pharmaceutical composition for the prevention and / or treatment of cisplatin-induced acute kidney injury, or for the prevention and / or treatment of non-tumor diseases associated with ferroptosis.

[0011] Preferably, the dosage form of the pharmaceutical composition is tablets, capsules, powders, granules, injections, or oral liquids.

[0012] Preferably, the phenazine-1-carboxylic acid prevents and / or treats cisplatin-induced acute kidney injury by inhibiting ferroptosis.

[0013] More preferably, the phenazine-1-carboxylic acid effectively inhibits ferroptosis under cystine deprivation conditions.

[0014] The fourth aspect of the present invention discloses that the above-mentioned pharmaceutical composition further includes use in combination with one or more therapeutic agents, said therapeutic agents being selected from chemotherapeutic drugs, antioxidants, anti-inflammatory drugs or iron chelators.

[0015] The fifth aspect of the present invention discloses the use of the above-described pharmaceutical composition in the preparation of a medicament for treating and / or preventing acute kidney injury caused by cisplatin chemotherapy.

[0016] The sixth aspect of the present invention discloses the use of the above-described pharmaceutical composition in the preparation of a medicament for treating and / or preventing non-tumor diseases associated with ferroptosis.

[0017] Compared with the prior art, the beneficial effects of this invention are as follows:

[0018] 1. This invention is the first to discover and demonstrate that phenazine-1-carboxylic acid can effectively alleviate cisplatin-induced acute kidney injury. In a cisplatin-induced acute kidney injury model in mice, intraperitoneal injection of phenazine-1-carboxylic acid effectively protects renal function, specifically by significantly reducing serum urea nitrogen and creatinine levels; alleviating pathological damage to kidney tissue and lowering renal tubular injury scores; and inhibiting the expression of inflammatory factors and damage markers in kidney tissue. Its therapeutic effect is comparable to that of the classic ferroptosis inhibitor ferrostatin-1, providing a new candidate drug for addressing the dose-limiting toxicity of cisplatin in clinical applications.

[0019] 2. In vitro cell experiments demonstrated that, under RSL3-induced or cystine deprivation-triggered ferroptosis conditions, phenazine-1-carboxylic acid effectively inhibited the death of HT1080 and MEF cells, reduced the accumulation of lipid peroxidation products, and decreased lactate dehydrogenase (LDH) and ATP leakage in cell culture supernatants. In vivo experiments further confirmed that phenazine-1-carboxylic acid treatment reduced the level of malondialdehyde (MDA), the end product of lipid peroxidation, in the kidney tissue of cisplatin-based mice, thus validating its nephroprotective effect at the molecular level by inhibiting the key pathway of ferroptosis.

[0020] 3. Phenazine-1-carboxylic acid, as a structurally well-defined compound, exhibits stable chemical properties. Animal experiments have shown that significant therapeutic effects can be achieved through conventional intraperitoneal injection, suggesting good in vivo activity. Given the central role of ferroptosis in various pathological processes, the application of phenazine-1-carboxylic acid disclosed in this invention is not limited to cisplatin-induced kidney injury but extends to other non-neoplastic diseases associated with ferroptosis, such as ischemia / reperfusion injury, neurodegenerative diseases, and drug-induced liver / kidney injury, providing new potential drug options for the prevention and treatment of these diseases.

[0021] 4. The pharmaceutical compositions of this invention are designed with in mind the possibility of combination use with other therapeutic agents such as chemotherapeutic drugs, antioxidants, anti-inflammatory drugs, or iron chelators. This lays the foundation for developing more optimized and synergistic treatment strategies in clinical practice to enhance renal protection or simultaneously manage tumors and other complications. Attached Figure Description

[0022] Figure 1 The results are experimental findings used to identify phenazine-1-carboxylic acid as an inhibitor of ferroptosis through high-throughput screening.

[0023] Figure 2 To investigate the inhibitory effect of phenazine-1-carboxylic acid on ferroptosis under cystine deprivation conditions.

[0024] Figure 3 and Figure 4 This study demonstrates the in vivo therapeutic effect of phenazine-1-carboxylic acid on cisplatin-induced acute kidney injury (AKI) in mice. Detailed Implementation

[0025] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but this does not limit the present invention to the scope of the described embodiments. Process parameters not specified in the embodiments of this application can be performed according to conventional methods, and all raw materials used can be obtained through commercial channels. Some experimental steps can be performed according to the corresponding product instructions.

[0026] Example 1: High-throughput screening and identification of phenazine-1-carboxylic acid as an inhibitor of ferroptosis (Figure).

[0027] HT1080 cells were seeded at a density of 10,000 cells per well in 96-well plates. After 12 hours, RSL3 (ferroptosis inducer; working concentration 0.2 μM, final concentration) and a small molecule natural product library (containing 3651 small molecules; MedChemExpress (MCE), catalog number HY-L021) (working concentration 10 μM) or ferrostatin-1 (working concentration 10 μM) were added to the cell culture medium. Cells were then cultured for another 24 hours. Subsequently, Hoechst 33342 (MCE, catalog number HY-15559; 1 μg / mL) and propidium iodide (PI, 2 μg / mL) were used to label the nucleus and cell death, respectively. A high-content quantitative cell imaging system (CellInsight CX7 Pro) was used to quantify cell death. HCS) data analysis, using ferroptosis inhibitor ferstatin-1 as a positive control, selected phenazine-1-carboxylic acid as the research subject. Phenazine-1-carboxylic acid showed significant cytoprotective activity, with a protective rate comparable to ferstatin-1, and was preliminarily identified as a candidate ferroptosis inhibitor. Figure 1 As shown in A and B.

[0028] To further confirm that phenazine-1-carboxylic acid is an inhibitor of ferroptosis, further validation was conducted at the cellular level. HT1080 and MEF cells were treated with RSL3 (working concentration 1 μM), phenazine-1-carboxylic acid (working concentration 20 μM), or ferstatin-1 (working concentration 10 μM), and cell death was detected 6 hours after treatment. Figure 1(C), treated for 4 hours and then lipid peroxidation was detected. Figure 1 (D). Ferraphobia causes cell membrane rupture, leading to the leakage of cell contents, including LDH and ATP, into the extracellular environment. Therefore, detecting LDH and ATP levels in the cell culture medium can indirectly reflect ferroptosis. HT1080 and MEF cells were treated with RSL3 (working concentration 1 μM), phenazine-1-carboxylic acid (working concentration 20 μM), or ferstatin-1 (working concentration 10 μM), and extracellular LDH levels were measured. Figure 1 (E) and ATP levels ( Figure 1 (Middle F).

[0029] Example 2: Phenazine-1-carboxylic acid significantly inhibited ferroptosis under cystine deprivation conditions.

[0030] Under cystine deprivation conditions, HT1080 (14 h) and MEF (12 h) cells were treated with phenazine-1-carboxylic acid (working concentration 20 μM) or ferstatin-1 (working concentration 10 μM), and cell death was detected by flow cytometry. Figure 2 (A). Under cystine deprivation conditions, HT1080 (12 h) and MEF (10 h) cells were treated with phenazine-1-carboxylic acid (working concentration 20 μM) or ferstatin-1 (working concentration 10 μM), and lipid peroxidation was detected by flow cytometry. Figure 2 (B) In addition, under cystine deprivation experimental conditions, HT1080 (14 h) and MEF (12 h) cells were treated with phenazine-1-carboxylic acid (working concentration 20 μM) or ferstatin-1 (working concentration 10 μM). Cell culture supernatants were collected, centrifuged at 1000 rpm for 5 min, and transferred to new Eppendorf tubes for later use. Extracellular LDH levels were detected using the lactate dehydrogenase cytotoxicity assay kit (C0016) from Beyotime Biotechnology Co., Ltd. The experimental results are as follows: Figure 2 As shown in Figure C. Under cystine deprivation conditions, HT1080 (14 h) and MEF (12 h) cells were treated with phenazine-1-carboxylic acid (working concentration 20 μM) or ferstatin-1 (working concentration 10 μM). Cell culture supernatants were collected, centrifuged at 1000 rpm for 5 min, and transferred to new Eppendorf tubes for later use. Extracellular ATP levels were detected using the enhanced ATP assay kit (S0027) from Beyotime Biotech. The experimental results are shown in Figure C. Figure 2 As shown in D.

[0031] Example 3: Phenazine-1-carboxylic acid significantly alleviated cisplatin-mediated acute kidney injury.

[0032] 1. Experimental procedure for establishing a mouse model of acute kidney injury

[0033] Male C57BL / 6 mice, 6-8 weeks old and weighing 20-25 grams, were kept in a specific pathogen-free room at 22-23°C under a 12-hour light / dark cycle with free access to food and water. Experimental animals were grouped as follows: ① Control group; ② Cisplatin model group; ③ Phenyrazine-1-carboxylic acid treatment group; ④ Ferrostir-1 positive control group. Model establishment and drug administration:

[0034] 2. At the end of the experiment, a representative mouse from each group was photographed, and the results are shown in Figure 3B.

[0035] 3. At the end of the experiment, the mice were dissected and their kidneys were removed. One representative kidney from each group was photographed, and the results are shown in Figure 3C.

[0036] 4. At the end of the experiment, the body weight of each group of mice was recorded, as shown in Figure 3D.

[0037] The body weight of mice in the cisplatin model group was significantly lower than that in the control group (P<0.001); while the body weight loss of mice in the phenazine-1-carboxylic acid treatment group and the ferstatin-1 positive control group was significantly reduced, and the difference was statistically significant compared with the cisplatin model group (P<0.001), indicating that phenazine-1-carboxylic acid can alleviate the weight loss of mice caused by cisplatin and improve the overall nutritional status and toxicity.

[0038] 5. At the end of the experiment, the mice were dissected, their kidneys were removed and weighed, and the results are shown in Figure 3E.

[0039] The kidney weight of mice in the phenazine-1-carboxylic acid treatment group and the ferstatin-1 positive control group was significantly lower than that in the cisplatin model group (P<0.001), indicating that phenazine-1-carboxylic acid can inhibit cisplatin-induced renal edema and reduce the degree of renal tissue damage.

[0040] 6. Based on the mouse's body weight and kidney weight, the ratio of kidney weight to body weight was calculated, and the result is shown in F of Figure 3.

[0041] The kidney-to-body weight ratio in the cisplatin model group was significantly higher than that in the control group, reflecting an increase in relative kidney volume, consistent with the pathological characteristics of acute kidney injury. The ratio in the phenazine-1-carboxylic acid treatment group and the ferstatin-1 positive control group was significantly lower than that in the cisplatin model group, further confirming that phenazine-1-carboxylic acid can effectively improve cisplatin-induced pathological kidney enlargement.

[0042] 7. Blood was collected from mice and incubated overnight at 4°C. The mice were then centrifuged at 2000 rpm for 10 minutes. Serum was collected to detect blood urea nitrogen levels using a kit (C013-2-1) from Nanjing Jiancheng Biotechnology Institute. The results are shown in Figure 3 (G).

[0043] In the control group, serum urea nitrogen levels remained within the normal range; in the cisplatin model group, serum urea nitrogen levels were significantly elevated, indicating severe renal impairment; in the phenazine-1-carboxylic acid treatment group, serum urea nitrogen levels were significantly lower than those in the cisplatin model group, and the effect was comparable to that in the ferstatin-1 positive control group, indicating that phenazine-1-carboxylic acid can effectively protect renal function and reduce the accumulation of urea nitrogen in the body.

[0044] 8. Blood was collected from mice and incubated overnight at 4°C. The mice were then centrifuged at 2000 rpm for 10 minutes. Serum creatinine levels were measured using a kit (C011-2-1) from Nanjing Jiancheng Biotechnology Institute. The results are shown in Figure 3, H.

[0045] Creatinine is a core indicator of renal function. The serum creatinine level in the cisplatin model group was significantly higher than that in the control group, confirming the impairment of renal tubular filtration function. The serum creatinine level in the phenazine-1-carboxylic acid treatment group was significantly lower than that in the cisplatin model group, further verifying the protective effect of phenazine-1-carboxylic acid on cisplatin-induced renal function damage.

[0046] 9. Mouse kidney tissue was collected, ground, and prepared into a protein solution. The malondialdehyde (MDA) level was then measured, and the results are shown in Figure 3I.

[0047] Malondialdehyde (MDA) is the end product of lipid peroxidation, and its elevated levels are an important marker of ferroptosis. In the cisplatin-treated mouse model group, MDA levels in kidney tissue were significantly higher than in the control group, suggesting that cisplatin induces severe lipid peroxidation and ferroptosis in kidney tissue. In the phenazine-1-carboxylic acid treatment group, MDA levels in kidney tissue were significantly lower than in the cisplatin-treated group, and similar to the effect in the ferstatin-1 positive control group, indicating that phenazine-1-carboxylic acid exerts a renal protective effect by inhibiting lipid peroxidation and blocking the ferroptosis pathway.

[0048] 10. mRNA was extracted from mouse kidney tissue and converted into cDNA. The transcriptional levels of IL-6, Tnfα, Ngal, Kim-1, and IL-1β were detected by qRT-PCR. The results are shown in Figure 3 (J).

[0049] IL-6, Tnfα, and IL-1β are key inflammatory factors, while Ngal and Kim-1 are specific biomarkers of kidney injury. The transcriptional levels of these genes in the kidney tissue of mice in the cisplatin model group were significantly higher than those in the control group, indicating severe inflammatory response and renal tubular damage. The transcriptional levels of all genes in the phenazine-1-carboxylic acid treatment group were significantly lower than those in the cisplatin model group, suggesting that phenazine-1-carboxylic acid can inhibit cisplatin-induced renal inflammatory response, reduce the expression of kidney injury biomarkers, and alleviate renal tubular epithelial cell damage.

[0050] 11. Mouse kidney tissue was collected and stained with hematoxylin and eosin. The results are shown in Figure 4 (K).

[0051] In the control group, the renal tubular structure of the mice was intact, the epithelial cells were neatly arranged, and there was no degeneration, necrosis, or inflammatory cell infiltration. In the cisplatin model group, the renal tubular structure was severely damaged, with epithelial cell swelling and shedding, luminal narrowing or occlusion, interstitial congestion and edema, and a large number of inflammatory cell infiltrations. The renal tubular structural damage was significantly reduced in the phenazine-1-carboxylic acid treatment group and the ferstatin-1 positive control group, the epithelial cells were basically neatly arranged, and the inflammatory cell infiltration was significantly reduced, suggesting that phenazine-1-carboxylic acid can protect the integrity of the renal tubular structure and reduce histopathological damage.

[0052] 12. Mouse kidney tissue was collected and stained with periodic acid-Schiff stain. The results are shown in Figure 4, L.

[0053] Periodic acid-Schiff staining is mainly used to visualize the renal tubular basement membrane and glycogen content. In the control group, the renal tubular basement membrane of mice was continuous and intact, and stained evenly. In the cisplatin model group, the renal tubular basement membrane was broken and discontinuous, with uneven staining depth, indicating severe basement membrane damage. In the phenazine-1-carboxylic acid treatment group and the ferstatin-1 positive control group, the continuity of the renal tubular basement membrane was basically restored, and the staining was even. This indicates that phenazine-1-carboxylic acid can repair the cisplatin-damaged renal tubular basement membrane and maintain the structural stability of kidney tissue.

[0054] 13. Mouse kidney tissue was collected and stained with F4 / 80. The results are shown in M ​​in Figure 4.

[0055] F4 / 80 is a macrophage-specific marker, and a positive staining signal indicates the degree of macrophage infiltration. Only a small number of F4 / 80 positive cells were observed in the kidney tissue of the control group; the number of F4 / 80 positive cells was significantly increased and their distribution was widespread in the kidney tissue of the cisplatin model group, suggesting extensive macrophage infiltration and a severe inflammatory response; the number of F4 / 80 positive cells was significantly reduced in the phenazine-1-carboxylic acid treatment group and the ferstatin-1 positive control group, indicating that phenazine-1-carboxylic acid can inhibit macrophage infiltration into kidney tissue and alleviate local inflammatory response.

[0056] 14. According to Figure 4 K and Figure 4 The results of L were used to statistically analyze the renal tubular scores of mice, and the results are shown in Figure 4 (N).

[0057] The renal tubular injury score in the phenazine-1-carboxylic acid treatment group was significantly lower than that in the cisplatin model group, with mild to moderate injury predominating. There was no significant difference in score between the group and the ferstatin-1 positive control group, which quantitatively confirmed the alleviating effect of phenazine-1-carboxylic acid on renal tubular pathological injury.

[0058] 15. According to Figure 4 The results of the M-test were used to quantify the level of macrophages in kidney tissue, and the results are shown in Figure 4 (O).

[0059] Quantitative analysis of the area of ​​F4 / 80 staining positive regions was performed using ImageJ software. The level of macrophages in the kidney tissue of the cisplatin model group was significantly higher than that of the control group. The level of macrophages in the phenazine-1-carboxylic acid treatment group was significantly lower than that in the cisplatin model group and comparable to that in the ferstatin-1 positive control group, further verifying the mechanism by which phenazine-1-carboxylic acid reduces renal inflammation by inhibiting macrophage infiltration.

[0060] This invention is not limited to the above-described embodiments. Any changes in shape or structure are within the scope of protection of this invention. The scope of protection of this invention is defined by the appended claims. Those skilled in the art can make various changes, modifications, substitutions, combinations, and simplifications to these embodiments without departing from the principles and essence of this invention. All such changes and simplifications should be considered equivalent substitutions and fall within the scope of protection of this invention.

Claims

1. Use of phenazine-1-carboxylic acid in the preparation of medicaments for the prevention and / or treatment of cisplatin-induced acute kidney injury.

2. Use of phenazine-1-carboxylic acid in the preparation of medicaments for the prevention and / or treatment of non-tumor diseases associated with ferroptosis.

3. The application according to claim 2, characterized in that, The non-neoplastic diseases associated with ferroptosis include ischemia / reperfusion injury, neurodegenerative diseases, drug-induced liver injury, or drug-induced kidney injury.

4. A pharmaceutical composition, characterized in that, The drug comprises a therapeutically effective amount of phenazine-1-carboxylic acid and a pharmaceutically acceptable carrier, and is used for the prevention and / or treatment of cisplatin-induced acute kidney injury, or for the prevention and / or treatment of non-neoplastic diseases associated with ferroptosis.

5. The pharmaceutical composition according to claim 4, characterized in that, The dosage form of the pharmaceutical composition is tablets, capsules, powders, granules, injections, or oral liquids.

6. The pharmaceutical composition according to claim 4 or 5, characterized in that, The phenazine-1-carboxylic acid prevents and / or treats cisplatin-induced acute kidney injury by inhibiting ferroptosis.

7. The pharmaceutical composition according to claim 6, characterized in that, The phenazine-1-carboxylic acid effectively inhibits ferroptosis under cystine deprivation conditions.

8. The pharmaceutical composition according to any one of claims 4-7, characterized in that, The pharmaceutical composition may also be used in combination with one or more therapeutic agents selected from chemotherapeutic drugs, antioxidants, anti-inflammatory drugs, or iron chelators.

9. Use of the pharmaceutical composition of any one of claims 4 to 8 in the preparation of a medicament for treating and / or preventing acute kidney injury caused by cisplatin chemotherapy.

10. Use of the pharmaceutical composition of any one of claims 4 to 8 in the preparation of a medicament for treating and / or preventing non-tumor diseases associated with ferroptosis.

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