Application of senkyunolide I in preparation of medicine for preventing and treating organ injury and / or cytotoxicity induced by cis-platinum

Abstract

The invention discloses application of senkyunolide I in preparation of a medicine for preventing and treating organ injury and / or cytotoxicity induced by cis-platinum, and belongs to the field of medicines, and the mass fraction of the senkyunolide I is greater than or equal to 90%; through a large number of tests, the inventor finds that senkyunolide I can reduce cytotoxicity induced by cis-platinum and improve cell activity; the kidney injury caused by cisplatin chemotherapy is relieved, and the renal tubule injury caused by cisplatin is relieved; the liver injury caused by cisplatin chemotherapy is relieved, the activity of glutamic oxalacetic transaminase and glutamic-pyruvic transaminase induced by cisplatin is reduced, and the degeneration and necrosis of liver cells caused by cisplatin are relieved; moreover, the senkyunolide I can be orally administered, so that the safety risk and adverse reaction caused by the conventional amifostine injection administration can be solved, and the senkyunolide I is expected to be developed into a cisplatin chemotherapy adjuvant drug which can be orally administered.

Description

[0001] The present invention relates to the field of medical technology, and in particular to use of ligusticum lactone I in preparing drugs for preventing and treating cisplatin-induced organ damage and / or cytotoxicity. Background Art

[0002] Platinum-based anticancer drugs, represented by cisplatin, remain widely used clinically as chemotherapeutic agents. Cisplatin possesses a broad anticancer spectrum and strong antitumor activity, showing promising antitumor effects against a variety of tumors, including lung, cervical, head and neck, testicular, and ovarian cancers. However, cisplatin can cause serious adverse reactions, such as nephrotoxicity, hepatotoxicity, ototoxicity, and neurotoxicity, limiting its clinical application. The kidneys and liver are known to be major metabolic organs in the human body. Damage to these vital organs can lead to unpredictable and serious consequences, even potentially life-threatening.

[0003] Amifostine is currently used clinically as an adjuvant chemotherapy drug. Preemptive administration of amifostine mitigates organ damage caused by chemotherapy drugs such as cisplatin. It primarily inhibits the binding of chemotherapy drugs to DNA, reducing their cytotoxicity to normal tissues. However, amifostine can only be administered by injection, making it less convenient for clinical use. Furthermore, the potential risks associated with the production, storage, and use of injectable formulations are higher than those of oral formulations. Amifostine may also be associated with adverse reactions such as vomiting, hypotension, drowsiness, and sneezing. A small number of patients experience decreased serum calcium levels and decreased urinary cyclic adenosine monophosphate excretion, thus limiting its clinical application. To date, the specific molecular mechanisms underlying acute kidney injury and liver injury induced by platinum-based anticancer drugs such as cisplatin remain incompletely understood, and oral preventive and treatment options with satisfactory efficacy and safety are still lacking.

[0004] Broader activity screening and efficacy evaluation of natural ingredients derived from traditional Chinese medicine in order to discover safe, effective and orally administrable candidate drugs is an effective way to address the above-mentioned problems with the use of injectable amifostine, so as to better meet the clinical needs of cancer chemotherapy patients. Summary of the Invention

[0005] The purpose of the present invention is to provide the use of ligusticum lactone I in the preparation of drugs for preventing and treating cisplatin-induced organ damage and / or cytotoxicity, so as to solve the above problems.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: use of ligusticum lactone I in the preparation of drugs for preventing and treating cisplatin-induced organ damage and / or cytotoxicity.

[0007] Senkyunolide I (SI) is a natural phthalide compound found primarily in plants of the genera Angelica and Ligusticum, both in the Apiaceae family. These include the Chinese medicinal herbs Angelica sinensis, Chuanxiong, and Ligusticum chuanxiong. Naturally occurring senkyunolide I is a racemic isomer consisting of a pair of optical isomers in a 1:1 ratio. Its chemical structure is shown in the figure below:

[0008] .

[0009] As a preferred technical solution: the mass fraction of the ligusticum lactone I is ≥90%.

[0010] The ligusticum lactone I used in the present invention can be prepared according to the method described in the applicant's previous authorized patent ZL201711082169.6, such as Example 1.

[0011] Existing studies have shown that ligusticum lactone I has multiple pharmacological effects, including antioxidant, anti-inflammatory, anticoagulant, anti-atherosclerotic, migraine relief, and reduction of ischemia-reperfusion injury.

[0012] The inventors of this application have found through extensive experiments that:

[0013] Yangchuanolitone I can reduce cisplatin-induced cytotoxicity, especially renal cell toxicity, and improve cell viability.

[0014] Yangchuanxiong lactone I can alleviate kidney damage caused by cisplatin chemotherapy, especially reduce the renal tubular damage caused by cisplatin.

[0015] Yangchuanxiong lactone I can alleviate liver damage caused by cisplatin chemotherapy, reduce the activity of cisplatin-induced aspartate aminotransferase and alanine aminotransferase, and reduce liver cell degeneration and necrosis caused by cisplatin.

[0016] Compared with the existing technology, the advantages of the present invention are: oral administration of lignanolide I can alleviate cisplatin-induced organ damage and reduce cytotoxicity, and lignanolide I has good safety and is expected to be developed into an orally administered cisplatin chemotherapy adjuvant drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Comparison of changes in serum creatinine and urea nitrogen, indicators of renal injury in mice after modeling (compared with the cisplatin model group, *P<0.05, **P<0.01);

[0018] Figure 2 Pathological image of mouse kidney tissue stained with HE;

[0019] Figure 3 Comparison of changes in the activities of aspartate aminotransferase and alanine aminotransferase, indicators of liver damage in mice after modeling (compared with the cisplatin model group, **P<0.01);

[0020] Figure 4 Pathological image of mouse liver tissue stained with HE;

[0021] Figure 5 Comparison of changes in HK-2 cell viability after cisplatin stimulation (compared with the cisplatin model group, *P<0.05, ***P<0.001; compared with the cisplatin + 40μM LIG group, ## P<0.01; compared with the cisplatin + 20μM LIG group, &&P<0.01);

[0022] Figure 6 Schematic diagram of the dosage regimen in Example 1. DETAILED DESCRIPTION

[0023] The present invention will be further described below with reference to the accompanying drawings.

[0024] Example 1 Establishment of Cisplatin-Induced Organ Model and Drug Treatment

[0025] (1) Experimental animals: 36 SPF-grade C57 male healthy mice, weighing 20-25 g, purchased from Spefoc (Beijing) Biotechnology Co., Ltd., animal license number: SCXK (Beijing) 2024-0001.

[0026] (2) Experimental cells: Human renal cortical proximal tubule epithelial cells (HK-2 cells) were purchased from Wuhan Punosai Company.

[0027] (3) Reagents and drugs: Cisplatin was purchased from GlpBio, USA; creatinine (Cr) assay kit, urea nitrogen (BUN) test kit, aspartate aminotransferase assay kit, and alanine aminotransferase test kit were all purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.; Amifostine for injection was purchased from Merro Pharmaceutical Co., Ltd.; Cell activity and cell proliferation assay kit (CellCounting Kit-8, CCK8) was purchased from Biosharp, USA; Ligustilide (LIG) was purchased from Chengdu Mansite Biotechnology Co., Ltd.

[0028] (4) Model establishment and drug treatment: C57 mice were fed adaptively in the animal room for one week and then randomly divided into a control group, a cisplatin model group, an SI treatment group (three doses: 20 mg / kg, 40 mg / kg, and 80 mg / kg, corresponding to the "low dose group", "medium dose group", and "high dose group"), and a positive drug treatment group (200 mg / kg of amifostine), with 6 mice in each group. According to the literature (Steury MD, et al. Biochemical Journal. 2017, 474(14): 2301-2313.), the mice in the SI treatment group were first gavaged with each dose for one week, and the mice in the control group were gavaged with an equal amount of normal saline; on the fourth day of SI administration, the mice in the model group and the treatment group were intraperitoneally injected with 20 mg / kg of cisplatin, and the mice in the control group were intraperitoneally injected with an equal amount of normal saline. The mice in the positive control drug treatment group were intraperitoneally injected with 200 mg / kg of amifostine only 0.5 h before the injection of cisplatin. The dosing schedule is as follows: Figure 6 shown.

[0029] Regarding the above model establishment and drug treatment plan, it should be noted that:

[0030] (1) The current clinical method for preventing and treating cisplatin-induced acute organ damage is preventive administration. This is because the injection time of cisplatin is controllable. Generally, patients will be injected with amifostine in advance before chemotherapy to achieve the purpose of protecting organs. Therefore, the SI of the present invention also refers to this method and adopts preventive administration;

[0031] (2) The administration time of the SI group and amifostine was different based on the consideration of dosage. The dosage of amifostine was 200 mg / kg. In the inventors’ preliminary experiments, it was found that when SI administration was performed three days before sampling, there was almost no therapeutic effect; however, when SI administration was performed seven days (one week) before sampling, the therapeutic effect was significant.

[0032] In addition, according to the existing technology and the experiments of this application, the ability to administer the drug orally is a major advantage of SI. SI has good safety, and there was no death after the IV administration of SI at 900 mg / kg.

[0033] Example 2 Biological Sample Collection and Detection

[0034] (1) Tissue sample collection: No food or water for 24 hours before sampling. After 72 hours of intraperitoneal injection of cisplatin, the eyeballs were anesthetized and blood was collected. After standing at room temperature for 2 hours, the blood was centrifuged at 4°C and 3000r for 20 minutes. The serum was collected and stored at -20°C for later use. After blood collection, all mice were killed by cervical dislocation. The kidneys and livers of the mice were collected and washed in normal saline, dried with filter paper, and part of the kidneys and livers were immersed in 4% (w / v) paraformaldehyde for HE staining to observe tissue pathological changes; the remaining kidneys were stored at -80°C for later use.

[0035] (2) Biochemical index testing: Serum creatinine, urea nitrogen, aspartate aminotransferase activity, and alanine aminotransferase activity were tested. Serum and urine samples were centrifuged at room temperature at each time point, and the supernatant was aspirated. The corresponding test kit instructions were followed to calculate the corresponding numerical results.

[0036] (3) HE staining of tissues: Mouse liver and kidney tissues were fixed with 4% paraformaldehyde and then stained with HE. Microscope observation and photography were performed. Three locations were selected for each HE staining slide. Pathological sections were used to observe liver and kidney damage.

[0037] Example 3: Cnidium monnieri Lactone I Alleviates Renal Damage Induced by Cisplatin Chemotherapy

[0038] (1) Evaluation of renal injury in mice: 24 hours after modeling, compared with the control group, the urea nitrogen and blood creatinine levels of mice in the cisplatin group were significantly increased (P<0.01); compared with the cisplatin group, the urea nitrogen levels of mice in the SI treatment group (20, 40, 80 mg / kg) and the amifostine treatment group were significantly decreased (P<0.05), and the blood creatinine levels of mice in the medium and high dose SI treatment groups (40, 80 mg / kg) and the amifostine treatment group were significantly decreased (P<0.01). The results are shown in Figure 1 , Figure 1 n=6; A: urea nitrogen; B: serum creatinine; compared with the cisplatin group: *P<0.05, **P<0.01.

[0039] (2) Pathological changes in mouse renal tissue: 24 hours after modeling, pathological examination showed that the renal cell structure of the control group mice was intact, and the glomeruli and renal tubules were intact. However, the kidneys of the mice in the cisplatin group showed obvious renal tubular damage, which was manifested by thinning of the cytoplasm, loss of the brush border, vacuolar necrosis of renal tubular cells, and a large number of gelatin casts in the renal tubules. The renal tissue pathology of mice in the SI (40, 80 mg / kg) and amifostine treatment groups was significantly improved. Results are shown in Figure 2 , Figure 2 Middle, ×200; black arrows: vacuolar necrosis of renal tubular cells; green arrows: numerous gelatinous casts in the renal tubules.

[0040] Example 4: Cnidium monnieri Lactone I Alleviates Liver Damage Induced by Cisplatin Chemotherapy

[0041] (1) Evaluation of liver damage in mice: 24 hours after modeling, the activities of aspartate aminotransferase and alanine aminotransferase in the cisplatin group mice were significantly increased compared with those in the control group (P<0.01); compared with the cisplatin group mice, the aspartate aminotransferase activities in the SI treatment group (20, 40, 80 mg / kg) and the positive control drug amifostine treatment group were significantly decreased (P<0.01), and the alanine aminotransferase activities in the medium and high dose SI treatment groups (40, 80 mg / kg) and the amifostine treatment group were significantly decreased (P<0.01). The results are shown in Figure 3 , Figure 3 n=6; A: AST activity; B: ALT activity; compared with the cisplatin group: **P<0.01.

[0042] (2) Pathological changes in mouse liver tissue: 24 hours after modeling, pathological examination showed that the liver cell structure of the mice in the control group was intact, while the liver cells of the mice in the cisplatin group showed obvious degeneration and necrosis, which was manifested by the increase in liver cell volume and the cytoplasm filled with small vacuoles. The mice in the SI treatment group and the amifostine treatment group showed liver cell renewal and regeneration, and the liver tissue pathology was significantly improved, which was manifested by the condensation of multiple liver cell nuclei and the presence of several binuclear liver cells, indicating that the liver cell renewal and regeneration ability was strong. The results are shown in Figure 4 , Figure 4 Middle, ×200; black arrows: hepatocyte degeneration and necrosis; yellow arrows: hepatocyte regeneration.

[0043] Example 5: Cnidium monnieri Lactone I Alleviates Cytotoxicity Induced by Cisplatin Chemotherapy

[0044] (1) Cell viability assay: According to the literature (Bunel V, et al. Toxicol In Vitro. 2015; 29(3): 458-467.), the density of HK-2 cells was adjusted to 1×10 5 Cells were seeded in 96-well plates at 100 μL / well and incubated at 37°C in a 5% CO2 incubator for 24 hours. Cells were divided into control, cisplatin-stimulated, and drug-treated groups (SI-10 μM, SI-20 μM, SI-40 μM, LIG-10 μM, LIG-20 μM, and LIG-40 μM) and cultured for another 24 hours. Cisplatin and drug-treated groups were administered simultaneously, while the control group received serum-free medium. After cell treatment, 10 μL of CCK8 solution was added to each well of the 96-well plate and cultured for another hour. Absorbance was measured at 450 nm.

[0045] (2) Changes in HK-2 cell viability: After 24 hours of cisplatin stimulation of HK-2 cells, CCK-8 results showed that the cell viability of the cisplatin group decreased significantly (P<0.001), while SI (20, 40 μM) administration and incubation significantly improved the viability of cisplatin cells (P<0.05); compared with the LIG administration group at the same concentration, the cell viability of the SI administration group (20, 40 μM) increased significantly (P<0.01), indicating that the cell protective effect of ligustilide I is stronger than that of the phthalide component ligustilide with similar structure. The results are shown in Figure 5 , Figure 5 n=5; compared with the cisplatin group: *P<0.05, ***P<0.001; compared with the SI-40 μM group: ##P<0.01; compared with the SI-20 μM group: &&P<0.01.

[0046] Example 6 Acute toxicity test

[0047] SPF-grade ICR mice, half male and half female, were purchased from Chengdu Dashuo Laboratory Animal Co., Ltd. (Laboratory Animal Production License No. SCXK(Chuan)2020-030). The test drug was injected via the tail vein (300 mg / time, three times daily), and any toxic reactions were observed and recorded.

[0048] The results showed that no mice died on the day of injection with lignanolide I. Over 14 days of observation, lignanolide I had no significant effect on the weight gain of mice compared to the blank group, and there was no significant difference in weight change compared to the blank group (P>0.05). Therefore, the maximum tolerable dose of lignanolide I is >900 mg / kg / day.

[0049] It should be noted that:

[0050] The experimental results of the above examples were statistically analyzed and quantified using GraphPad Prism V8.0 software. The data were expressed as x ± s. All data were in accordance with the normal distribution. The t-test was used for comparison between two groups, and one-way analysis of variance was used for comparison between multiple groups. P < 0.05 indicated that the difference was statistically significant.

[0051] 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 and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of ligusticum lactone I in the preparation of drugs for preventing and treating cisplatin-induced organ damage and / or cytotoxicity.

2. The use according to claim 1, characterized in that: The mass fraction of the ligusticum chuanxiong lactone I is ≥90%.

3. The use according to claim 1, characterized in that: The organ is the liver.

4. The use according to claim 3, characterized in that: The liver damage is hepatocyte degeneration and necrosis.

5. The use according to claim 1, characterized in that: The organ is the kidney.

6. The use according to claim 5, characterized in that: The kidney damage is renal tubular damage.

7. The use according to claim 1, characterized in that: The cytotoxicity is renal cytotoxicity.

Images