Medical application of O-demethylangorapin

Abstract

The invention provides an application of O-demethylangorapin in preparation of a medicine for relieving renal toxicity and myotoxicity of cis-platinum and cachexia manifestation caused by the renal toxicity and the myotoxicity of the cis-platinum. The active ingredient of the medicine is one or more of O-demethylated angorapin, a hydrate of the O-demethylated angorapin, a pharmaceutically acceptable salt of the O-demethylated angorapin, a tautomer of the O-demethylated angorapin, a stereoisomer of the O-demethylated angorapin and a precursor compound of the O-demethylated angorapin. The O-demethylangiecarpin has the potential to be developed into a drug for relieving the toxicity of cis-platinum.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to application of O-desmethylangoracin in the preparation of a drug for alleviating cisplatin nephrotoxicity and myotoxicity. Background Art

[0002] Cisplatin is a widely used and broad-spectrum chemotherapy drug suitable for the treatment of various cancers, including lung cancer, ovarian cancer, bladder cancer, head and neck tumors, and various digestive tract tumors. However, cisplatin also has many toxic side effects, such as nephrotoxicity, skeletal muscle toxicity, neurotoxicity, and bone marrow suppression, which seriously limit its clinical application. These toxic side effects make many cancer patients unable to tolerate chemotherapy containing cisplatin, and cause many complications, weakening the therapeutic effect of cisplatin and seriously affecting the quality of life of patients. Currently, there is a lack of methods to reduce the toxicity of cisplatin and related complications, and the main method is hydration before cisplatin administration. Cisplatin can directly damage the renal tubules and directly affect drug metabolism, limiting the use of subsequent drugs. About 30% of patients will develop acute kidney injury after cisplatin chemotherapy, and even lead to death, and more than 60% of patients with acute kidney injury will develop chronic kidney disease (Manohar S, Leung N. Cisplatin nephrotoxicity: a review of the literature. J Nephrol, 2018. 31(1): 15-25.). The skeletal muscle atrophy caused by cisplatin can seriously affect the mobility of cancer patients and increase the risk of accidents such as falls. In the elderly cancer patient population, the decreased balance ability caused by skeletal muscle atrophy may directly threaten life. Currently, there is a lack of clinical treatment measures to reduce cisplatin nephrotoxicity and skeletal muscle atrophy. There is a great need for the prevention and treatment of kidney damage and skeletal muscle atrophy caused by cisplatin. Seeking methods or therapeutic drugs to reduce cisplatin toxicity can improve the efficacy of cisplatin-based chemotherapy regimens, increase the treatment benefits of cancer patients, and protecting renal function can also continuously guarantee patients' various medication needs.

[0003] Currently, the core pathological basis of cisplatin-induced renal injury is that drug accumulation in renal tubular cells directly causes DNA damage and mitochondrial damage, leading to PTEC death, which in turn causes renal inflammation and renal fibrosis (Tang C, Livingston MJ, Safirstein R, Dong Z. Cisplatin nephrotoxicity: new insights and therapeutic implications. Nat Rev Nephrol, 2023. 19(1): 53-72.). In recent years, many natural medicines have been shown to be effective in preventing and treating cisplatin nephrotoxicity and improving the therapeutic benefits of cisplatin (Zhang QY, Wang FX, Jia KK, et al. Natural Product Interventions for Chemotherapy and Radiotherapy-Induced Side Effects. Front Pharmacol, 2018. 9: 1253.). Cisplatin can cause symptoms such as anorexia and skeletal muscle atrophy in various cancer models. Cisplatin-related muscle atrophy is mainly related to inflammatory response and protein degradation (Sakai H, Sagara A, Arakawa K, et al. Mechanisms ofcisplatin-induced muscle atrophy. Toxicol Appl Pharmacol. 2014; 278(2): 190-199.). Co-culture of mouse C2C12 myotube cells with cisplatin causes atrophy of C2C12 myotube cells, activates the ubiquitin-proteasome pathway, and further causes protein degradation (Sakai H, Ikeno Y, Tsukimura Y, et al. Upregulation of ubiquitinated proteins and their degradation pathway in muscle atrophy induced by cisplatin in mice. Toxicol Appl Pharmacol. 2020;403: 115165.). The pharmacological effects of small molecule compounds on reducing cisplatin toxicity can be preliminarily evaluated by applying the small molecule compounds to the above in vitro and in vivo models. Summary of the Invention

[0004] The present invention aims to solve the problems in the prior art and provides O-desmethylangolensin (O-desmethylangolensin, ODMA, C 15H 14 O4) in the preparation of a drug for reducing cisplatin nephrotoxicity and myotoxicity and its effects on body weight and food intake.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides the use of O-demethylangoracin in the preparation of a drug for reducing the nephrotoxicity and myotoxicity of cisplatin, wherein the active ingredient of the drug is one or more of O-demethylangoracin, a hydrate of O-demethylangoracin, a pharmaceutically acceptable salt of O-demethylangoracin, a tautomer of O-demethylangoracin, a stereoisomer of O-demethylangoracin, and a precursor compound of O-demethylangoracin.

[0007] Preferably, the O-desmethylangoracin is the only active ingredient of the drug.

[0008] The second aspect of the present invention provides the use of O-demethylangoracin in the preparation of a drug for alleviating cachexia symptoms caused by cisplatin, wherein the active ingredient of the drug is one or more of O-demethylangoracin, a hydrate of O-demethylangoracin, a pharmaceutically acceptable salt of O-demethylangoracin, a tautomer of O-demethylangoracin, a stereoisomer of O-demethylangoracin, and a precursor compound of O-demethylangoracin.

[0009] Preferably, the O-desmethylangoracin is the only active ingredient of the drug.

[0010] The present invention adopts the above technical solution, which has the following technical effects compared with the prior art:

[0011] The present invention's research results show that ODMA has no significant proliferation inhibitory effect on mouse C2C12 myoblasts, C2C12 myotubes, and human HK2 renal tubular epithelial cells after 24, 48, and 72 hours of in vitro exposure, but has a stronger proliferation inhibitory effect on mouse lung cancer LLC cells. ODMA can alleviate cisplatin-induced atrophy of C2C12 myotubes, which is related to ODMA inhibiting cisplatin-induced skeletal muscle protein degradation. ODMA can also reduce cisplatin-induced DNA damage in HK2 cells, alleviating cisplatin nephrotoxicity. In in vivo experiments, ODMA can alleviate the symptoms of cachexia caused by cisplatin, such as weight loss, decreased food intake, skeletal muscle weight loss, kidney weight loss, and epididymal fat weight loss; at the same time, it can downregulate the mRNA expression levels of ubiquitin ligases Fbxo32 and Trim63 in the ubiquitin-proteasome pathway in skeletal muscle, reduce skeletal muscle protein degradation in the ubiquitin-proteasome pathway, and reduce the reduction of muscle fiber cross-sectional area; at the same time, it can reduce the kidney damage index, lower blood urea nitrogen and creatinine levels, protect renal function, and reduce renal toxicity.

[0012] In summary, ODMA can inhibit the ubiquitin-proteasome pathway to reduce protein degradation, alleviate muscle atrophy, reduce cellular DNA damage directly caused by cisplatin, reduce nephrotoxicity, and generally improve cachexia symptoms in mouse models. It has the potential to be prepared as a drug to reduce the toxicity of cisplatin. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 In one embodiment of the present invention, different concentrations of ODMA were used to treat mouse C2C12 myoblasts ( Figure 1 A), C2C12 myotube cells ( Figure 1 B), HK2 human renal tubular epithelial cells ( Figure 1 C), LLC mouse lung cancer cells ( Figure 1 D) Graph showing the impact of vitality;

[0014] Figure 2 This is a graph showing the effect of different concentrations of ODMA on the atrophy of C2C12 myotube cells treated with cisplatin in one embodiment of the present invention;

[0015] Figure 3 This is a graph showing the effects of different concentrations of ODMA on DNA damage in HK2 cells stimulated by cisplatin in one embodiment of the present invention;

[0016] Figure 4 The daily body weight ( Figure 4 A), tumor volume ( Figure 4 B) Food intake ( Figure 4 D) and final tumor weight ( Figure 4 C) Impact results diagram;

[0017] Figure 5 The effect of different concentrations of ODMA and the positive control drug Daidzein (DAI) on the gastrocnemius muscle weight of LLC tumor-bearing mice treated with cisplatin in one embodiment of the present invention ( Figure 5 A) Forelimb grip strength ( Figure 5 B) Epididymal fat weight ( Figure 5 C), kidney weight ( Figure 5 D), serum urea nitrogen ( Figure 5 E), creatinine ( Figure 5 F) Horizontal impact results diagram;

[0018] Figure 6 This figure shows the effect of ODMA on the cross-sectional area of ​​gastrocnemius muscle fibers in LLC tumor-bearing mice treated with cisplatin in one embodiment of the present invention;

[0019] Figure 7 This is a graph showing the effect of ODMA on the renal tubular injury index of LLC tumor-bearing mice treated with cisplatin in one embodiment of the present invention;

[0020] Figure 8 This figure shows the effect of ODMA on the mRNA expression of ubiquitin ligases Fbxo32 and Trim63 in the gastrocnemius muscle of LLC tumor-bearing mice treated with cisplatin in one embodiment of the present invention. DETAILED DESCRIPTION

[0021] The present invention provides the use of ODMA in the preparation of a drug for reducing the toxicity of cisplatin. The active ingredient of the drug is one or more of ODMA, a hydrate of ODMA, a pharmaceutically acceptable salt of ODMA, a tautomer of ODMA, a stereoisomer of ODMA, and a precursor compound of ODMA. The drug also includes a pharmaceutically acceptable carrier or excipient.

[0022] The term "pharmaceutically acceptable salt" refers to a salt formed by the compound with a pharmaceutically acceptable inorganic acid or organic acid, the inorganic acids including but not limited to hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, and sulfuric acid; the organic acids including but not limited to formic acid, acetic acid, propionic acid, succinic acid, 1,5-naphthalenedisulfonic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethylacetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methanesulfonic acid, p-toluenesulfonic acid, citric acid, and amino acids; the term "pharmaceutically acceptable" refers to a substance that is suitable for use in humans without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, a substance with a reasonable benefit / risk ratio.

[0023] The term "tautomer" refers to functional group isomers resulting from the rapid shift of an atom between two positions in a molecule, for example, an enol and the corresponding ketone.

[0024] The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, such as cis-trans isomers, enantiomers, conformers, and the like.

[0025] The term "precursor compound" refers to a compound that is inactive in vitro but can be converted into the active ingredient of the present invention through metabolism or chemical reactions in the body, thereby exerting its pharmacological effect.

[0026] The present invention will be described in detail and specifically below through specific examples to provide a better understanding of the present invention, but the following examples do not limit the scope of the present invention.

[0027] Example 1

[0028] This example provides an experiment to explore the inhibitory effect of ODMA on the viability of mouse C2C12 myoblasts, mouse C2C12 myotube cells, HK2 human renal tubular epithelial cells, and LLC mouse lung cancer cells. The specific experimental content and results are as follows:

[0029] Mouse C2C12 myoblasts, mouse C2C12 myotubes, human renal tubular epithelial HK2 cells, and mouse lung cancer LLC cells were co-cultured with different concentrations of ODMA for 24 h, 48 h, and 72 h, and cell viability was detected using the CCK8 assay. The results are shown in Figure 2. Figure 1 As shown:

[0030] The inhibition rate of ODMA on each cell line at 24 h, 48 h, and 72 h did not reach 50%, and the overall proliferation inhibition rate was not high. Among them, the inhibition rate of ODMA on mouse C2C12 myoblasts, mouse C2C12 myotubes, and human renal tubular epithelial HK2 cells at 24 h, 48 h, and 72 h was too low, and the IC50 Unable to calculate ( Figure 1 A, B, C). The inhibition rate of ODMA on LLC cells at 24 h and 48 h was too low, IC 50 Unable to calculate, IC at 72 h 50 437.5 μM ( Figure 1 D). ODMA has a relatively strong inhibitory effect on the viability of mouse lung cancer LLC cells.

[0031] Example 2

[0032] This example verifies the effect of ODMA on alleviating atrophy of mouse C2C12 myotube cells treated with cisplatin (DDP). The specific experimental content and results are as follows:

[0033] Mouse C2C12 myoblasts were induced to differentiate into myotubes with 2% horse serum. C2C12 myotubes treated with 40 μM DDP were treated with gradient concentrations of ODMA (5 μM, 10 μM, 20 μM) for 24 h. After fixation, permeabilization, and blocking, the cells were incubated with MyHC primary antibody at 4°C overnight. After discarding the primary antibody, the cells were washed and incubated with the same type of fluorescent secondary antibody for 2 hours. After washing, the cells were incubated with DAPI for 5 minutes for nuclear staining. After adding anti-fluorescence quencher, fluorescence microscopy was used to photograph the cells. The results are shown in Figure 3. Figure 2 As shown. Figure 2 The MyHC-positive area and the number of nuclei inside and outside the myotubes were counted, and the percentage of fluorescent area and the fusion index (the ratio of the number of nuclei inside the myotube to the total number of nuclei in the same field of view) were calculated. After 24 hours of DDP treatment, C2C12 myotubes significantly decreased in area, atrophied, and exhibited a decreased fusion index. Following ODMA treatment, the area of ​​MyHC-labeled myotubes gradually increased with increasing ODMA concentrations, and the fusion index increased, indicating that ODMA can alleviate DDP-induced atrophy in C2C12 myotubes (*P < 0.05, **P < 0.01, ***P < 0.001 compared with the model group).

[0034] Example 3

[0035] This example verifies the protective effect of ODMA on human renal tubular epithelial HK2 cells stimulated by cisplatin. The specific experimental content and results are as follows:

[0036] HK2 cells were collected at 2×10 5The cells were plated in 6 cm culture dishes at a density of 1000 cells / dish, and C2C12 myotube cells treated with 40 μM DDP were intervened with gradient concentrations of ODMA (5 μM, 10 μM, 20 μM) for 24 h. After fixation, permeabilization, and blocking, the cells were incubated with γ-H2AX primary antibody at 4°C overnight. After discarding the primary antibody, the cells were washed and incubated with the same type of fluorescent secondary antibody for 2 hours. After washing again, the cells were incubated with DAPI for 5 minutes for cell nucleus staining. After adding anti-fluorescence quencher, fluorescence microscope photos were taken. The results are shown as follows: Figure 3 As shown in the figure, cells with positive γ-H2AX expression were counted and the percentage of positive cells was calculated. A higher proportion of γ-H2AX-positive cells appeared in HK2 cells after DDP treatment, while gradient concentrations of ODMA reduced the proportion of positive cells, indicating that the proportion of cells with DNA damage decreased. ODMA can significantly alleviate DDP-induced DNA damage in HK2 cells in a dose-dependent manner (compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001).

[0037] Example 4

[0038] This example verifies the effect of ODMA on the alleviation of nephrotoxicity, skeletal muscle toxicity and cachexia-related symptoms and signs in LLC tumor-bearing mice treated with cisplatin. The specific experimental content and results are as follows:

[0039] LLC cells (2 × 10 6On the first day, 36 C57BL / 6 mice were subcutaneously inoculated on the right upper back. The 36 tumor-bearing mice were divided into six groups, each consisting of six mice: a tumor control group (LLC group), a cisplatin-treated group (LLC+DDP group), a low-dose ODMA combined treatment group (LLC+DDP+ODMA(L) group), a medium-dose ODMA combined treatment group (LLC+DDP+ODMA(M) group), a high-dose ODMA combined treatment group (LLC+DDP+ODMA(H) group), and a positive drug DAI treatment group (LLC+DDP+DAI group). Six C57 mice from the same batch served as a normal control group (NC group). On the fifth day, when the tumors reached a long diameter of 5 mm, drug administration began. Cisplatin was administered by intraperitoneal injection, and ODMA and DAI were both prepared as suspensions in 0.5% sodium carboxymethylcellulose and administered by oral gavage. Dosage regimen: NC group (0.5% sodium carboxymethyl cellulose), LLC group (0.5% sodium carboxymethyl cellulose), LLC+DDP group (DDP 4 mg / kg), LLC+DDP+ODMA (L) group (DDP 4 mg / kg + ODMA 10 mg / kg), LLC+DDP+ODMA (M) group (DDP 4 mg / kg + ODMA 20 mg / kg), LLC+DDP+ODMA (H) group (DDP 4 mg / kg + ODMA 40 mg / kg), cisplatin administration cycle is once every 3 days, ODMA is once a day. Body weight, tumor size, body weight, tumor size and food intake were measured before each administration. DDP can significantly reduce the body weight of mice ( Figure 4 A), inhibition of tumor volume and final tumor weight ( Figure 4 B, C), reduce food intake ( Figure 4 D, E) ODMA intervention can alleviate the effects of cisplatin on body weight and food intake, but has no effect on the tumor inhibitory effect of DDP (compared with LLC+DDP group, *P<0.05, **P<0.01, ***P<0.001).

[0040] On the 18th day, all mice were sacrificed and samples were collected to measure tumor weight, gastrocnemius muscle weight, kidney weight, and epididymal fat weight. The forelimb grip strength was tested before sacrifice. The results showed that after ODMA intervention, the gastrocnemius muscle weight of mice ( Figure 5 A) Forelimb grip strength ( Figure 5 B) Epididymal fat weight ( Figure 5 C), kidney weight ( Figure 5 D) significantly improved (compared with LLC+DDP group, *P<0.05, **P<0.01, ***P<0.001). Serum urea nitrogen and creatinine levels were tested. ODMA can reduce the increase of urea nitrogen and creatinine caused by cisplatin and improve renal function ( Figure 5E, F). H&E staining of gastrocnemius muscle sections showed that the cross-sectional area of ​​gastrocnemius muscle fibers in mice was significantly increased after ODMA intervention. Figure 6 As shown (compared with LLC+DDP group, **P<0.01, ***P<0.001). H&E staining of kidney sections showed that ODMA could reduce the renal tubular injury index ( Figure 7 ) (Compared with the LLC+DDP group, *P<0.05, **P<0.01, ***P<0.001). Gastrocnemius muscle was collected and qPCR was used to detect the mRNA expression of ubiquitin ligases Fbxo32 and Trim63. ODMA can downregulate the expression of Fbxo32 and Trim63. Figure 8 As shown (*P < 0.05, **P < 0.01, ***P < 0.001 compared with the LLC + DDP group).

[0041] From the above examples, it can be seen that ODMA can reduce kidney damage and skeletal muscle atrophy caused by cisplatin, alleviate cachexia symptoms, and does not affect the anti-tumor effect of cisplatin, which provides a strategy for preparing drugs that reduce the toxic side effects of cisplatin.

[0042] While the specific embodiments of the present invention have been described in detail above, these are merely exemplary and the present invention is not limited thereto. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, any equivalent changes and modifications made without departing from the spirit and scope of the present invention are intended to be encompassed within the scope of the present invention.

Claims

1. The use of O-demethylangoracin in the preparation of a drug for reducing cisplatin nephrotoxicity and myotoxicity, characterized in that: The active ingredient of the drug is one or more of O-demethylangoracin, a hydrate of O-demethylangoracin, a pharmaceutically acceptable salt of O-demethylangoracin, a tautomer of O-demethylangoracin, a stereoisomer of O-demethylangoracin, and a precursor compound of O-demethylangoracin.

2. The use according to claim 1, characterized in that The O-desmethylangoracin is the only active ingredient of the drug.

3. The use of O-desmethylangoracin in the preparation of a drug for alleviating cachexia symptoms caused by cisplatin, characterized in that: The active ingredient of the drug is one or more of O-demethylangoracin, a hydrate of O-demethylangoracin, a pharmaceutically acceptable salt of O-demethylangoracin, a tautomer of O-demethylangoracin, a stereoisomer of O-demethylangoracin, and a precursor compound of O-demethylangoracin.

4. The use according to claim 3, characterized in that The O-desmethylangoracin is the only active ingredient of the drug.

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