CN120131630A - Use of orlistat in preparing drugs for preventing and treating cisplatin-induced renal injury - Google Patents

Abstract

The invention belongs to the technical field of medicines, and discloses application of orlistat in preparation of a medicament for preventing and treating cisplatin-induced kidney injury. The invention discloses an application of orlistat or pharmaceutically acceptable salt thereof in preventing and/or treating kidney injury for the first time. In vitro cell experiments show that orlistat obviously inhibits toxicity of cisplatin to tubular epithelial cells, inhibits DNA damage, reduces ROS generation, improves endoplasmic reticulum stress and inhibits apoptosis. Animal experiments show that orlistat can obviously improve toxicity of cisplatin to mice, increase survival rate of mice, restore weight of mice, improve kidney pathological injury, inhibit secretion of inflammatory factors, reduce creatinine, urea nitrogen and reverse electrolyte disorder. In particular, orlistat does not affect the anti-tumor efficacy of cisplatin at the cellular level and in tumor-bearing mice, and the protective effect of cisplatin-induced kidney injury is superior to that of the sole clinical drug amifostine.

Description

Application of orlistat in preparation of medicines for preventing and treating cisplatin-induced kidney injury

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an application of orlistat in preparing a medicine for preventing and treating cisplatin-induced kidney injury.

Background

Cisplatin, also known as cis-dichloro-diammine platinum, was used clinically since 1969 and was approved by the FDA in 1978 for the treatment of testicular cancer. Cisplatin is the first platinum antitumor drug widely used in clinic and is also a representative drug of the first platinum. Because of the advantages of high anticancer activity, wide antitumor spectrum, less cross drug resistance and the like, the preparation method has become one of basic drugs for chemotherapy of various solid tumors since acquisition. Even though the current targeted drugs, immunotherapy and other emerging antitumor drugs are widely applied, cisplatin is still widely applied clinically as a first-line basic therapeutic drug or an auxiliary therapeutic drug for lung cancer, ovarian cancer, osteosarcoma, head and neck tumor, gastric cancer and the like, and is even the preferred combination drug for some advanced malignant tumors. Cisplatin is statistically nearly 50% used in chemotherapy of malignant tumors.

The anti-tumor mechanism of cisplatin is clear, namely after cisplatin enters cells, due to the reduction of chloride ion concentration, cisplatin is dissociated and directly combined with DNA to form intra-strand or inter-strand crosslinking of DNA, thereby interfering the replication of DNA and causing irreversible damage and death of the cells. Although cisplatin has good antitumor effects, there is also greater toxicity, common and serious toxicities including nephrotoxicity, ototoxicity, electrolyte disorders, and the like.

Although the second and third generation platinum such as carboplatin and oxaliplatin have significantly reduced nephrotoxicity, neurotoxicity and bone marrow toxicity have significantly increased. Therefore, in clinical applications, the third generation platinum species each have different clinical indications and application ranges. Cisplatin remains one of the irreplaceable classical chemotherapeutics for many malignant tumors for over five decades, but its kidney damage greatly limits its clinical application.

Nephrotoxicity is a major dose limiting factor in cisplatin clinical administration. Among patients who are clinically treated with cisplatin, up to 30% of patients develop acute kidney injury (Acute Kidney Injury, AKI). Of those that do not meet AKI criteria after cisplatin treatment, up to 70% of those have a risk of developing Chronic kidney disease (Chronic KIDNEY DISEASE, CKD). Thus, patients with tumors often face the risk of acute and chronic kidney injury when treated with cisplatin.

Cisplatin is not absorbed by oral administration, and is clinically administered by intravenous administration. The transport proteins of the basolateral membrane and the luminal lateral membrane of tubular cells are responsible for uptake and efflux of cisplatin. Cisplatin is taken up into tubular epithelial cells via organic cation transporter2 (Organic Cation Transporter, OCT 2), organic anion transporters 1 and 3 (Organic Anion Transporter/3, oat 1/3), etc., and the concentration of cisplatin accumulated in proximal tubular epithelial cells can reach 5 times that in blood. Cisplatin, even at blood concentrations that are non-toxic, may reach toxic levels in the kidneys. Cisplatin entering cells induces DNA damage, oxidative stress, endoplasmic reticulum stress, cell cycle arrest, etc., and eventually causes inflammation, apoptosis. Based on differences in cisplatin dose and frequency, nephrotoxicity may exhibit AKI and/or renal interstitial fibrosis. A single high dose of cisplatin results in rapid death of a large number of tubular epithelial cells, resulting in acute loss of kidney function, producing AKI, and multiple doses of cisplatin result in tubular epithelial cell cycle arrest, myofibroblast activation, extracellular matrix deposition, resulting in renal interstitial fibrosis, producing chronic kidney injury.

In clinical practice, the most commonly used strategies for preventing/ameliorating cisplatin kidney injury are hydration regimens of physiological saline, hypertensive physiological saline, mannitol, furosemide, and the like. However, there is currently no uniform standard regimen for cisplatin hydration due to the definition of nephrotoxicity, the dose of cisplatin administered, and the differences in patient populations in different clinical trials. Furthermore, existing clinical trial results do not fully support existing clinical practices, e.g., renal protection of mannitol, combined with physiological saline treatment may increase the incidence of hyponatremia in patients.

The antioxidant/protectant amifostine (Amifostine, AMI) is the only drug approved by the us FDA for reducing renal toxicity in ovarian cancer and non-small cell lung cancer. In clinical practice, the recommended dose of amifostine is typically 910mg/m ², which is instilled by intravenous drip (completed within 15 minutes) 30 minutes prior to cisplatin administration. Because amifostine is mainly excreted via the kidneys, the use of high doses of amifostine requires careful consideration of drug concentration in clinical combination for patients with renal insufficiency. Meanwhile, amifostine may itself cause side effects such as hypotension, nausea, etc. Currently, some clinical trials attempt low dose regimens (e.g., 500mg/m ²) or divided doses, but have evidence of efficacy. The effects of amifostine on reversing cisplatin nephrotoxicity in the different experiments are very different, and the protection effect on cisplatin-induced kidney injury is not stable. Therefore, cisplatin nephrotoxicity protective drugs are a significant and urgent need in clinical anti-tumor practice.

The new use of old drugs, also called drug repositioning or drug repositioning, refers to drugs that are being clinically used for treating diseases, and candidate drugs that were or are in preclinical or clinical studies, and find new uses through various methods of research or practice. Compared with the large investment, long period and high risk of new drug research and development, the old drug new use reduces the risk of drug research and development failure, shortens the research and development period, reduces the research and development cost, improves the research and development success rate, and has incomparable advantages compared with 'new drug'. In the field of 'old medicine new use' for improving cisplatin damage, some progress is also made at present, for example, diphenhydramine has a certain protection effect on cisplatin nephrotoxicity in cell and animal experiments. Clinical test results show that the proton pump inhibitor pantoprazole can reduce the renal toxicity caused by cisplatin application to patients with head and neck tumors, but in the study of cisplatin application to patients with juvenile osteosarcoma, pantoprazole does not have obvious protective effect on cisplatin renal toxicity.

Orlistat is the first approved over-the-counter weight loss drug by FDA, developed by Hoffmann-LaRoche, switzerland, first marketed in new zealand in 1998, in the united states in 1999, and in china in 2000. Orlistat is a long-acting and powerful gastrointestinal lipase inhibitor, and its chemical structure is tetrahydrolipstatin derivative. Orlistat inhibits the hydrolysis of about 30% of triglycerides in the diet by covalently binding to the serine active sites of gastric and pancreatic lipases, reducing the absorption of free fatty acids and monoacylglycerols. Orlistat has no effect on amylase, trypsin, phospholipase A2, etc., and no effect on carbohydrate, protein and phospholipid absorption. Orlistat is not substantially absorbed after oral administration, and has low drug concentration in plasma, and plasma concentration <5ng/mL within 8 hours after oral administration of a single dose (maximum dose 800 mg). 97% of the drug is excreted by the feces, and a small amount (< 2%) of the drug in the original form and the metabolites are excreted from the kidneys. The orlistat has good safety and no in-vivo drug accumulation after long-term administration. The acute toxicity to mice, rats, pups and dogs is low by single oral administration, and the doses of 1000mg/kg (dogs), 2000mg/kg (rats) and 5000mg/kg (rats, mice) do not show toxic symptoms. The LD50 of the rat lavage is more than 5g/kg, and the rat lavage has no irritation, reproduction toxicity, carcinogenesis and mutagenesis effects. The orlistat has slight toxic and side effects, and the main adverse reactions of patients after oral administration are steatorrhea, abdominal discomfort, intestinal flatulence and the like.

GLP-1 weight-losing medicines such as liraglutide and semraglutide are appeared, so that the weight-losing market is thoroughly changed. GLP-1 drugs are favored, and the market is rapidly atrophic due to the embarrassing side effect of fatty diarrhea of orlistat. The orlistat manufacturers and products in China are numerous, and the subsequent competition is to further realize the white-heat treatment. How to cope with the real problem which is not acceptable for pharmaceutical enterprises. Thus, the development of new indications for orlistat that are not weight-reducing is helpful in achieving its secondary development.

Disclosure of Invention

Aiming at the current situation that the cisplatin nephrotoxicity of the basic anticancer chemotherapy drug lacks an effective prevention and treatment drug, the invention provides a new application of orlistat, namely the application of orlistat in preventing and/or treating cisplatin-induced kidney injury.

The object of the first aspect of the present invention is to provide the use of orlistat or a pharmaceutically acceptable salt thereof for the manufacture of a product for the prevention and/or treatment of renal injury.

The object of the second aspect of the present invention is to provide the use of orlistat or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the reduction of cisplatin nephrotoxicity.

The object of a third aspect of the present invention is to provide a medicament.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

In a first aspect of the invention there is provided the use of orlistat or a pharmaceutically acceptable salt thereof in the manufacture of a product for the prevention and/or treatment of renal injury.

In some embodiments of the invention, the kidney injury is a pharmaceutical kidney injury.

In some embodiments of the invention, the pharmaceutical kidney injury is an anti-tumor drug.

In some embodiments of the invention, the antineoplastic agent is a platinum antineoplastic agent.

In some embodiments of the invention, the platinum-based antineoplastic agent is cisplatin.

In some embodiments of the invention, the kidney injury comprises cisplatin-induced kidney injury.

In some embodiments of the invention, the cisplatin-induced kidney injury includes acute kidney injury, chronic kidney injury, and electrolyte disorders associated therewith.

In some embodiments of the invention, the kidney injury includes acute and chronic kidney injury and electrolyte disorders associated therewith caused by single high dose and/or multiple small dose cisplatin.

In some embodiments of the invention, the product achieves prevention and/or treatment of cisplatin-induced kidney injury by ameliorating, reversing, or reversing cisplatin-induced electrolyte disorders.

In some embodiments of the invention, the electrolyte disorder includes at least one of hypomagnesemia and hypocalcemia.

In some embodiments of the invention, the product achieves prevention and/or treatment of cisplatin-induced kidney injury by reducing the level of inflammatory factor expression.

In some embodiments of the invention, the inflammatory factors include TNF- α, IL-1β, and IL-6.

In some embodiments of the invention, the product achieves prevention and/or treatment of cisplatin-induced kidney injury by reducing creatinine, urea nitrogen.

In some embodiments of the invention, the product achieves prevention and/or treatment of cisplatin-induced kidney injury by reducing expression of KIM-1, pro-apoptotic proteins p53, CLEAVED CASPASE.

In some embodiments of the invention, the agent achieves prevention and/or treatment of cisplatin-induced kidney injury by inhibiting cisplatin-induced DNA damage and oxidative stress.

In some embodiments of the invention, the agent prevents and/or treats cisplatin-induced kidney injury by inhibiting cisplatin-induced epithelial cell death, apoptosis.

In some embodiments of the invention, the agent prevents and/or treats cisplatin-induced kidney injury by inhibiting cisplatin-induced epithelial endoplasmic reticulum stress and activation of MAPKs pathways.

Experiments prove that the orlistat can obviously inhibit toxicity of cisplatin-induced rat renal tubular epithelial NRK-52E cells, human proximal renal tubular epithelial HK-2 cells and primary cultured proximal renal tubular epithelial cells (PTEC), reduce Reactive Oxygen Species (ROS) generation, inhibit DNA damage, inhibit endoplasmic reticulum stress and inhibit apoptosis. At the same time, orlistat can reduce the death rate of normal mice induced by cisplatin, reduce the blood urea nitrogen and creatinine level, improve the pathological changes of kidneys, and obviously inhibit the nephrotoxicity of tumor-bearing mice, reduce the inflammatory factor level and reverse electrolyte disorder during treatment of tumor-bearing mice. The protective effect of orlistat on cisplatin nephrotoxicity is superior to the unique FDA-approved drug amifostine. Orlistat has no influence on the effect of cisplatin on killing malignant tumor cells in vitro, has no influence on the in vivo antitumor activity of cisplatin, and can even enhance the killing effect of cisplatin on tumors.

In some embodiments of the invention, the pharmaceutically acceptable salt comprises at least one of hydrochloride, hydrobromide, sulfate, phosphate, acetate, citrate, lactate, ascorbate, maleate, tartrate, malate, gluconate, sulfonate, citrate, benzoate, benzenesulfonate, carbonate, methanesulfonate, stearate, nitrate, valerate, or succinate.

In some embodiments of the invention, the effective dose of orlistat or a pharmaceutically acceptable salt thereof in the product is 10-50 μm.

In a second aspect of the invention there is provided the use of orlistat or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the reduction of cisplatin nephrotoxicity.

In some embodiments of the invention, the agent achieves the effect of reducing cisplatin nephrotoxicity by inhibiting cisplatin-induced DNA damage and oxidative stress (reducing cisplatin-induced ROS).

In some embodiments of the invention, the agent achieves the effect of reducing cisplatin nephrotoxicity by inhibiting cisplatin-induced epithelial cell death, apoptosis.

In some embodiments of the invention, the epithelial cells include tubular epithelial cells (e.g., NRK-52E, HK-2 and PTEC cells).

In some embodiments of the invention, the agent inhibits cisplatin-induced increase in p53, CLEAVED CASPASE expression, promoting expression of the anti-apoptotic protein Bcl-2.

In some embodiments of the invention, the agent achieves the effect of reducing cisplatin nephrotoxicity by inhibiting activation of cisplatin-induced epithelial endoplasmic reticulum stress (inhibiting CHOP and ATF-4 expression and phosphorylation of eif2α) and MAPKs pathway (inhibiting phosphorylation of Erk, JNK and p38 proteins).

In some embodiments of the invention, the pharmaceutically acceptable salt comprises at least one of hydrochloride, hydrobromide, sulfate, phosphate, acetate, citrate, lactate, ascorbate, maleate, tartrate, malate, gluconate, sulfonate, citrate, benzoate, benzenesulfonate, carbonate, methanesulfonate, stearate, nitrate, valerate, or succinate.

In some embodiments of the invention, the effective dose of orlistat or a pharmaceutically acceptable salt thereof in the medicament is 10-50 μm.

Orlistat or a pharmaceutically acceptable salt thereof can obviously inhibit toxicity of cisplatin to tubular epithelial cells, inhibit elevation of p53 protein and reduction of Bcl-2, inhibit cleavage of Caspase-3, inhibit activation of MAPKs, endoplasmic reticulum stress and reduce oxidative stress, and inhibit apoptosis of tubular epithelial cells.

In a third aspect of the invention there is provided a medicament comprising orlistat or a pharmaceutically acceptable salt thereof and cisplatin.

Orlistat obviously improves the general state of mice after cisplatin administration, obviously improves the survival rate of the mice, reduces the levels of blood creatinine, urea nitrogen and inflammatory factors, improves electrolyte disorder and obviously improves kidney injury. The protective effect of orlistat on cisplatin-induced kidney injury does not affect the in-vitro and in-vivo anti-tumor activity of cisplatin, and the protective effect is superior to that of the currently clinically used medicine amifostine.

In some embodiments of the invention, the medicament further comprises pharmaceutically acceptable excipients, such as at least one of solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, tonicity modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-binding agents, integrating agents, permeation promoters, pH adjusting agents, buffering agents, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retarders, carriers.

In some embodiments of the present invention, the active ingredient may be formulated with any one or more pharmaceutically acceptable excipients to a specific dosage form for ease of administration. Such adjuvants may be diluents (e.g., starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, microcrystalline cellulose, etc.), absorbents (e.g., calcium sulfate, calcium hydrogen phosphate, light magnesium oxide, and calcium carbonate, etc.), wetting agents (e.g., water, ethanol, etc.), binders (e.g., hypromellose, povidone, starch slurry, and syrup, etc.), disintegrants (e.g., dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants, and crospovidone, etc.), lubricants (e.g., magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol, and micronized silica gel, etc.), colorants (e.g., titanium dioxide, sunset, methylene blue, and pharmaceutically acceptable iron oxide, etc.), coating materials (e.g., acrylic resin, hypromellose, and povidone, etc.), solvents (e.g., water for injection, ethanol, propylene glycol, DMSO, and glycerin, etc.), acid-base regulators (e.g., hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid, and sodium tartrate, etc.), antioxidants (e.g., sodium carbonate, sodium pyro, and sodium thiosulfate, etc.), bacteriostats (e.g., phenol, sodium sulfite, sodium metabisulfite, and sodium chloride, etc.), and sodium chloride, etc.

In some embodiments of the invention, the dosage form of the product comprises a parenterally administered dosage form or a parenterally administered dosage form, preferably a parenterally administered dosage form.

In some embodiments of the invention, the parenteral dosage form comprises at least one of a powder, a tablet, a granule, a capsule, a sustained release formulation, a solution, a dry suspension, an effervescent tablet, an emulsion, a suspension, a syrup, a drop, a chewable tablet.

In some embodiments of the invention, the parenteral dosage form includes, but is not limited to, enteric coated tablets, film coated tablets, sugar coated tablets, dispersible tablets, sucking tablets, chewing tablets, effervescent tablets, scored tablets, sustained release coated tablets, controlled release tablets, orally disintegrating tablets, buccal tablets, and the like.

In some embodiments of the invention, the parenteral dosage form comprises at least one of an injectable dosage form, a respiratory dosage form, a dermal dosage form, a mucosal dosage form, a luminal dosage form.

In some embodiments of the present invention, the injectable administration form includes, but is not limited to, an injection, a solution for injection, an injection for intravenous drip, a suspension for injection, a sterile powder for injection, an intravenous injection, a water injection, an emulsion for injection, a powder injection, an injection, a sterile powder injection, a lyophilized powder injection, and the like.

The beneficial effects of the invention are as follows:

The invention discloses an application of orlistat or pharmaceutically acceptable salt thereof in preventing and/or treating kidney injury for the first time. In vitro cell experiments show that orlistat obviously inhibits toxicity of cisplatin to tubular epithelial cells, inhibits DNA damage, reduces ROS generation, improves endoplasmic reticulum stress and inhibits apoptosis. But orlistat has no inhibitory effect on cisplatin killing tumor cells. Animal experiments in vivo show that orlistat can remarkably improve survival rate of mice treated by cisplatin, restore weight of the mice, improve kidney pathological damage, inhibit secretion of inflammatory factors, reduce creatinine, urea nitrogen and reverse electrolyte disorder. In particular, orlistat does not affect the anti-tumor efficacy of cisplatin in vivo, and its kidney protecting effect is superior to that of the sole clinical drug amifostine. This suggests that orlistat or a pharmaceutically acceptable salt thereof is a good drug for the prevention and treatment of cisplatin nephrotoxicity. Orlistat is an FDA approved drug, has few side effects, high safety and great commercial value, is clinically applied for decades, and has good application prospect when being developed into a drug for protecting cisplatin nephrotoxicity.

Drawings

FIG. 1 is a graph showing that Orlistat (ORL) inhibits Cisplatin (CIS) -induced cytotoxicity of rat tubular epithelial cells NRK-52E. Wherein A is ORL (25. Mu.M) significantly reverses cisplatin-induced changes in cell morphology (left) and MTT assay (right), scale 400 μm, and B is Propidium Iodide (PI) staining, scale 400 μm. * Representing P <0.05.

FIG. 2 is an illustration of the inhibition of cisplatin-induced cytotoxicity of human proximal tubular epithelial cells HK-2 by Orlistat (ORL). Wherein A is ORL (25. Mu.M) significantly reverses cisplatin-induced changes in cell morphology (left) and MTT assay (right), scale 400 μm, and B is Propidium Iodide (PI) staining, scale 400 μm. * Representing P <0.05.

FIG. 3 is a graph showing that Orlistat (ORL) inhibits Cisplatin (CIS) -induced cytotoxicity of primary Proximal Tubular Epithelial Cells (PTEC) extracted from Balb/c mice. Wherein A is ORL (25. Mu.M) significantly reverses cisplatin-induced changes in cell morphology (left) and MTT assay (right), scale 400 μm, and B is Propidium Iodide (PI) staining, scale 400 μm. * Representing P <0.05.

Fig. 4 is an Orlistat (ORL) reversal of Cisplatin (CIS) -induced expression of the tubular epithelial apoptotic pathway protein. Wherein A, B, C is Western Blotting to detect cisplatin apoptosis pathway proteins P53, bcl-2, caspase-3 and CLEAVED CASPASE-3 in three cells of NRK-52E (A), HK-2 (B) and PTEC (C), respectively.

FIG. 5 is an illustration of the reverse Cisplatin (CIS) -induced apoptosis of rat tubular epithelial cells NRK-52E by Orlistat (ORL). Wherein A is the result of 7-AAD/annexin V flow double dyeing, B is the result of TUNEL dyeing, and the scale is 400 μm.

Fig. 6 is an Orlistat (ORL) reversal of Cisplatin (CIS) -induced production of reactive oxygen species (reactive oxygen species, ROS) and DNA damage in tubular epithelial cells. Wherein A is the result of detecting the DNA damage of HK-2 cells by comet electrophoresis, the amplification factor is 200, B is the result of detecting the flow cytometry of the ROS in NRK-52E cells by using a fluorescent probe DCFH ₂ -DA, and AMI is the clinical drug amifostine approved by FDA. CON was a blank control group and H ₂O₂ was a positive control group.

FIG. 7 is an Oligostat (ORL) reversal of Cisplatin (CIS) -induced activation of MAPKs pathways and endoplasmic reticulum stress pathways of the tubular epithelial cells NRK-52E. Wherein A is the expression of endoplasmic reticulum stress pathway protein, and B is the detection of MAPKs pathway protein expression by Western Blotting.

Fig. 8 is that Orlistat (ORL) does not affect the killing of malignant cells by Cisplatin (CIS). Wherein A-F are respectively the cytotoxicity of orlistat combined with cisplatin on triple-negative breast cancer 4T1 cells (A), non-small cell lung cancer A549 cells (B), colon cancer HCT116 cells (C), ovarian cancer SKOV3 cells (D), cervical cancer Hela cells (E) and large cell lung cancer H460 cells (F).

FIG. 9 is the effect of Orlistat (ORL) on Cisplatin (CIS) -induced nephrotoxicity in normal mice. Wherein A is a schematic diagram of animal experiment design flow, B is a survival curve graph of mice, and C is a weight statistical graph of mice during the experiment. In each figure, CON is a physiological saline group, MODEL is a cisplatin group singly administered, ORL-L is a low-dose orlistat combined cisplatin group, ORL-M is a medium-dose orlistat combined cisplatin group, ORL-H is a high-dose orlistat combined cisplatin group, and AMIFOSTINE is a positive drug amifostine combined cisplatin group.

FIG. 10 is the protective effect of Orlistat (ORL) on Cisplatin (CIS) induced kidney injury in mice bearing 4T1 tumors. Wherein A is a schematic diagram of animal experiment design flow, B is a tumor volume statistical graph of tumor-bearing mice, C is a weight statistical graph of tumor-bearing mice, D is an inter-group survival curve of tumor-bearing mice, E is a photograph of each group of tumors, F is a photograph of each group of kidneys, G is H & E staining results of each group of kidneys, and the scale is 500 μm. In the figure, CON is a physiological saline group, CIS is a cisplatin single treatment group, CIS+ORL-L is a low-dose orlistat combined cisplatin group, CIS+ORL-M is a medium-dose orlistat combined cisplatin group, CIS+ORL-H is a high-dose orlistat combined cisplatin group, and CIS+AMI is a positive drug amifostine combined cisplatin group.

FIG. 11 is the protective effect of Orlistat (ORL) on Cisplatin (CIS) induced kidney injury in mice bearing 4T1 tumors. Wherein A is blood urea nitrogen, creatinine, serum magnesium ion, serum calcium ion, serum TNF alpha, hemoglobin, renal tissue IL-1 beta and renal tissue IL-6 level, B is expression of kidney injury biomarker KIM-1, apoptosis protein p53, c-caspase-3 and caspase-3 protein detected by Western Blotting in kidney tissue of tumor-bearing mice. In the figure, ns represents no significant difference, P <0.05, P <0.01, and P <0.001.

Detailed Description

The following describes the present invention in further detail by way of specific examples.

It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Orlistat is a long-acting, potent inhibitor of gastrointestinal lipase, developed by roche company, and first available in 1998 for use in the treatment of obesity. Orlistat (Orlistat, ORL) (CAS number: 96829-58-2) has the chemical structure:

The experimental cell lines used in the examples were NRK-52E cells (rat renal tubular cell epithelial cells), HK-2 cells (human renal cortex proximal tubular epithelial cells), SKOV3 cells, A549 cells, HCT116 cells, heLa cells, H460 cells and 4T1 cells were all purchased from American ATCC cell banks, and the mouse renal tubular epithelial primary cells PTEC were isolated from the mouse kidneys and primary cultured.

Cisplatin used in the examples was purchased from Shanghai Ala Biotechnology Co., ltd. And amifostine trihydrate, a positive drug, was purchased from Shanghai Micin Biotechnology Co., ltd. Paraformaldehyde, xylene, H & E dye liquor, neutral gums were all purchased from bio-technology limited of beijing solebao.

Antibodies used in the examples, caspase 3 antibody (9662 s), p53 antibody (1C 12) and p-H2AX (20E 3) were purchased from CELL SIGNALING (CST), GAPDH antibody (60004-1-ig) and KIM-1 antibody (30948-1-AP) were purchased from Proteintech.

Cell culture NRK-52E cells were cultured with complete medium (DMEM medium containing 5% FBS and 1% diabody) and HK-2 cells were cultured with complete medium (mixed medium containing 10% FBS and 1% diabody DMEM/F-12:1). The culture was carried out under conditions of constant temperature of 37℃and 5% CO ₂.

Preparation of drug mother liquor, namely, taking orlistat (purchased from Shanghai Michelin Biochemical technology Co., ltd., purity > 99%) powder, weighing, dissolving with DMSO solution to prepare mother liquor with 25mM concentration, and taking cisplatin (purity > 99%) powder, weighing, and dissolving with DMSO solution to prepare mother liquor with 3mM concentration. All mother liquor was stored in a-20 ℃ refrigerator.

Example 1

Cisplatin (15. Mu.M) was administered 4 hours prior to ORL treatment in the NRK-52E cell line, and the cells were subjected to MTT cell viability assay and PI staining 24 hours later. The operation of this embodiment is specifically as follows:

Taking NRK-52E cells in logarithmic growth phase, culturing 5000 cell plates in a 96-well plate, and standing at 5% CO ₂ and 37 ℃ overnight for growth. After the cells were fully expanded and acclimatized to the plating environment, the complete medium was removed and transferred into medium containing 1% fbs. With or without administration of 50 μl of double strength Orlistat (ORL) medium solution (0 or 50 μΜ), and with or without administration of 50 μl of double strength cisplatin medium solution (0 or 30 μΜ) after 4 hours. The final concentration of ORL was 25. Mu.M and that of cisplatin was 15. Mu.M, both of which were administered to the cells in combination or alone. After 24 hours of treatment, cell viability was measured at 570nm using conventional MTT methods. In addition, after 24 hours of treatment, 20. Mu.L of Propidium Iodide (PI) (5 mg/mL) was given for incubation, and observed under a fluorescence microscope and photographed.

As shown in FIG. 1, after the NRK-52E cells are treated by cisplatin for 24 hours, the cell viability is reduced from 100% to about 50%, the ORL is not obviously influenced after 24 hours alone, the ORL and the cisplatin are combined for 24 hours, the cell viability is obvious and reaches about 80%, the cell morphology is obviously improved, and the cell number is obviously and normally increased. PI staining showed an increase in the number of PI positive cells (stained red) following cisplatin treatment, whereas the number of PI positive cells in the ORL pretreatment group was significantly decreased. This suggests that ORL may significantly protect cisplatin-induced NRK-52E cell death.

Example 2

Cisplatin (30. Mu.M) was administered 4 hours prior to ORL treatment in the HK-2 cell line, and the cells were subjected to MTT cell viability assay and PI staining 24 hours later. The specific operation of this example is as in example 1. The final concentration ORL was 25 μm and cisplatin was administered to the cells at 30 μm either in combination or alone.

As shown in FIG. 2, the cell viability of HK-2 cells after 24 hours of cisplatin treatment was reduced from 100% to about 50%, the cell viability was not significantly affected after 24 hours of ORL alone, the cell viability was significantly increased to about 80% after 24 hours of ORL and cisplatin combination administration, the cell morphology was significantly improved, and the cell number was also significantly increased normally. PI staining showed an increase in the number of PI positive cells (stained red) following cisplatin treatment, whereas the number of PI positive cells in the ORL pretreatment group was significantly decreased. This suggests that ORL may significantly protect cisplatin-induced HK-2 cell death.

Example 3

Cisplatin was administered 15 μm4 hours prior to ORL treatment in primary proximal tubular epithelial cells (PTEC cells) extracted from Balb/c mice, and cells were subjected to MTT cell viability assay and PI staining 24 hours later. This example operates as example 1. The final concentration was ORL 25. Mu.M and cisplatin 15. Mu.M were administered to the cells either in combination or alone.

As shown in FIG. 3, the cell viability of PTEC cells after 24 hours of cisplatin treatment was reduced from 100% to 45%, and the cell viability was not significantly affected after 24 hours of ORL alone, and the cell viability was significantly increased, the cell morphology was significantly improved, and the cell number was also significantly increased after 24 hours of ORL in combination with cisplatin. PI staining showed an increase in the number of PI positive cells (stained red) following cisplatin treatment, whereas the number of PI positive cells in the ORL pretreatment group was significantly decreased. This suggests that ORL may significantly protect cisplatin-induced PTEC cell death.

Example 4

Effect of ORL on cisplatin-induced expression of NRK-52E, HK-2 and PTEC apoptosis-related proteins. The administration mode of this example is the same as that of examples 1 to 3, and Western blotting detects apoptosis pathway proteins to examine the effect of ORL on cisplatin-induced apoptosis pathway of tubular epithelial cells, and the specific process is as follows:

NRK-52E, HK-2 cells in logarithmic growth phase and primary cultured PTEC cells were cultured in 6-well plates, respectively, and grown overnight at 5% co ₂ at 37 ℃. After the cells were fully expanded and acclimatized to the plating environment, the complete medium was removed and transferred to medium containing 1% FBS. After 4 hours without or with ORL 25. Mu.M, cells were collected and total protein was extracted and expression of apoptosis-related proteins (caspase 3 protein, CLEAVED CASPASE protein, p53 protein, bcl-2 protein) in each cell was examined by conventional Western blotting.

As shown in FIG. 4, cisplatin has no obvious effect on the total caspase 3 expression in three cells, and the induction of CLEAVED CASPASE is obviously increased, which suggests that cisplatin induces caspase 3-mediated apoptosis, that cisplatin induces the expression of pro-apoptotic protein p53 in NRK-52E cells to be obviously increased, that anti-apoptotic protein Bcl-2 to be obviously reduced, and that the expression of pro-apoptotic protein p53 in HK-2 cells to be obviously increased. ORL pretreatment significantly inhibited cisplatin-induced increases in p53 expression, CLEAVED CASPASE, and reversed decreases in Bcl-2 expression. This demonstrates that under the conditions of the examples, apoptosis is significantly induced after 24 hours of cisplatin stimulation, and that ORL has a significant protective effect on cisplatin-induced apoptosis of NRK-52E, HK-2 and PTEC.

Example 5

Cisplatin was administered at 15. Mu.M 4 hours prior to ORL treatment in the NRK-52E cell line (ORL and cisplatin treatment dose and experimental procedure were the same as in example 1), cells were collected after 24 hours, 7-AAD/annexin V double-probe staining was incubated and flow cytometry double staining results were performed.

Bright field and green field photography TUNEL staining results were performed using Incucyte. TUNEL staining, a terminal deoxynucleotidyl transferase mediated dUTP notch end labeling method, is based on DNA fragmentation during apoptosis. Upon cleavage of genomic DNA, the exposed 3' -OH can be catalyzed by terminal deoxynucleotidyl transferase (Terminal Deoxynucleotidyl Transferase, tdT) plus Fluorescein (FITC) -labeled dUTP (fluoroscein-dUTP) and can be detected by fluorescence microscopy or flow cytometry. TUNEL staining is one of the important techniques for determining apoptosis.

As shown in FIG. 5, in the 7-AAD/annexin V flow double-staining results, cisplatin induced significant early apoptosis (Q3 in FIG. 5A) with an apoptosis rate as high as 30.2%, and ORL significantly reduced the proportion of early apoptotic cells induced by cisplatin to 9.7%. In TUNEL staining, the TUNEL dye is induced to bind to the end of the broken DNA of the cell under the stimulation of cisplatin to generate fluorescence, in green field, ORL can obviously reduce the number of TUNEL positive cells induced by cisplatin, and in bright field, ORL can reduce the reduction of the number of cells induced by cisplatin and can obviously improve the cell morphology.

Example 6

This example examines the effect of ORL on cisplatin-induced DNA damage and oxidative stress (ROS), as follows:

NRK-52E cells were treated as in example 1, cells were collected after 24 hours, and were stained with comet electrophoresis to determine DNA damage, and H ₂O₂ was used as a control drug for inducing DNA damage. The electrophoresis solution and the lysate are self-prepared, and are subjected to ice bath electrophoresis at 20V voltage for 20min in a DNA electrophoresis tank, and after electrophoresis, the solution is washed by deionized water, and is dripped with 10 mu L of PI dye and photographed under a fluorescence microscope.

Cisplatin 15. Mu.M was administered 4 hours prior to ORL treatment in NRK-52E (treatment method and dose were the same as in example 1), cells were harvested after 30 minutes and the ROS probe DCFH ₂ -DA 10. Mu.M was incubated and the results of the flow cytometry assay were performed. Wherein Amifostine (AMI) is used as a positive control drug.

The results are shown in FIG. 6, which shows that the ORL can significantly reduce DNA tailing phenomenon (comet) caused by cisplatin, and the result of comet electrophoresis of NRK-52E cells suggests that the ORL can significantly protect cisplatin-induced DNA damage. In the flow results of ROS assay, cisplatin treatment for half an hour induced the production of large amounts of ROS in the cell (the flow peak was significantly shifted to the right compared to the CON group), ORL significantly reduced the ROS production induced by cisplatin, and the effect was superior to that of amifostine, suggesting that ORL could inhibit oxidative stress caused by cisplatin.

Example 7

This implementation was used to examine the effect of ORL on cisplatin-induced endoplasmic reticulum stress and MAPKs pathway in tubular epithelial cells, as follows:

Using NRK-52E cells, the mode of administration and the drug dose were the same as in example 1.Western blotting examined endoplasmic reticulum stress and MAPKs pathway biomarkers to investigate the effect of ORL on cisplatin-induced endoplasmic reticulum stress and MAPKs.

As shown in FIG. 7, cisplatin can significantly induce the increase of CHOP and ATF-4 protein expression and the increase of EIF2α phosphorylation in the endoplasmic reticulum stress pathway, and the increase of Erk, JNK and p38 protein phosphorylation in the MAPKs pathway. The ORL can obviously inhibit the increase of cisplatin-induced protein expression of the signal channels, which suggests that the ORL can obviously inhibit cisplatin-induced endoplasmic reticulum stress and activation of MAPKs channels.

Example 8

The example is used for examining the influence of ORL on the effect of cisplatin on killing malignant tumor cells in vitro, and is specifically as follows:

Malignant tumor cells such as SKOV3 cells, A549 cells, HCT116 cells, heLa cells, H460 cells and 4T1 cells are adopted. Each cell plate was grown overnight in 96-well plates at 5% CO ₂ at 37 ℃. After the cells were fully expanded and acclimatized to the plating environment, the complete medium was removed and transferred to medium containing 1% FBS. Cisplatin (15. Mu.M) was administered to each cell after 4 hours of ORL treatment (final concentration of ORL was 0 to 200. Mu.M depending on the cell type), and MTT cell viability was measured on the cells after 24 hours in the same manner as in example 1.

As shown in fig. 8, the individual ORL administration did not promote proliferation of each malignant cell, and the ORL concentration-dependently inhibited proliferation of malignant cells on a549 cells, HCT116 cells, and H460 cells. In the experimental group where ORL and cisplatin were co-administered, the ORL at different concentrations had no significant protective effect on cisplatin-induced malignant cell death, as opposed to in tubular epithelial cells, and the killing activity of cisplatin was significantly enhanced at slightly higher concentrations. This shows that ORL does not affect the antitumor drug activity of cisplatin in vitro.

Example 9

In this example, animal experiments were used to investigate the effect of ORL on cisplatin-induced kidney injury in normal mice, which were selected to investigate whether ORL inhibited the renal injury effects of cisplatin itself at the animal level, as follows:

SPF-class Balb/c mice (25 g.+ -.3 g, from the university of Australian health sciences) of 6-7 weeks of age were selected and adapted to the environment for one week, and then were caged and grouped as DAY 0. The mice were divided into CON group, MODEL group, ORL low dose group (ORL-L group), ORL medium dose group (ORL-M group), ORL high dose group (ORL-H group) and positive drug AMIFOSTINE group (i.e., positive drug amifostine), each group being 6 mice. The CON group only carries out intraperitoneal injection of equivalent physiological saline, the MODEL group carries out single intraperitoneal injection of cisplatin (15 mg/kg), and the ORL is injected once daily on the basis of single cisplatin injection of the ORL low, medium and high dosage groups, and the dosages are respectively 1mg/kg, 3mg/kg and 10mg/kg. The cisplatin working solution is injected into the abdominal cavity at 15mg/kg once, the orlistat working solution is injected once a day, and AMI FOSTINE groups of administration modes are the same as the ORL groups, namely, on the basis of single cisplatin injection, the cisplatin working solution is injected into the abdominal cavity at AMIFOSTINE times a day, and the dosage is 200mg/kg. Starting with DAY 1, mice body weight was recorded daily, and on DAY 10, the mind was recorded, and no drug was given to the mice. Euthanasia was performed after the body weight was recorded on day 15.

The results are shown in FIG. 9. The mice in the other groups had significantly lower body weight than the CON group, and the mice in the positive drug AMI or ORL group had higher body weight than the MODEL group in the later period. Meanwhile, the ORL-H group started to stabilize body weight and had a tendency to rise back at day 12. All mice in MODEL group died on day 7, all mice in ORL-L group and AMIFOSTINE group died on day 12, all mice in ORL-M group died on day 13, while the mice in ORL-H group survived to the end of the experiment except one on day 10, and the body weight of the mice in ORL-H group showed a significant trend back at the end of the experiment. These results suggest that ORL can significantly combat nephrotoxicity of cisplatin at high single doses, significantly prolong survival curves and inhibit weight loss in mice, and that its effect is superior to that of amifostine, a positive drug.

Example 10

This example utilizes animal experiments to investigate the effect of ORL on cisplatin-induced kidney injury in tumor-bearing mice.

Because cisplatin is used as a basic chemotherapeutic drug, it is widely used in the treatment of various malignant tumors. One of the necessary prerequisites for the application of ORL as a means of inhibiting and protecting its renal injury is that ORL does not impair the antitumor efficacy of cisplatin. Therefore, in this example, xenograft tumor models of triple negative breast cancer 4T1 cells were selected to examine whether ORL would affect the anti-tumor efficacy of cisplatin. In clinical cisplatin chemotherapy, the treatment is generally carried out for 2-3 weeks, one cycle is carried out every week, and cisplatin is given 2-3 times in each cycle. The embodiment adopts a similar scheme with clinical cisplatin, and the specific scheme is as follows:

After SPF-grade Balb/c mice (25 g+ -3 g, from Zhuhai Bai Tong Biotechnology Co., ltd.) of 6-7 weeks old are selected to be suitable for environmental feeding for one week, all the mice are injected subcutaneously with 1X 10 ⁶ 4T1 cells a week in advance for molding. Random cage grouping was performed after tumor molding and was designated DAY 1. Tumor-bearing mice were grouped into CON, cisplatin (CIS), orlistat low, medium and high dose (CIS+ORL-L, CIS +ORL-M, CIS +ORL-H) and amifostine (CIS+AMI). The CON group only carries out intraperitoneal injection of equivalent physiological saline once a day, the CIS group carries out intraperitoneal injection of cisplatin 5mg/kg once every 3 days, the low, medium and high dose groups of orlistat are respectively administered by intraperitoneal injection of ORL 1mg/kg, 3mg/kg and 10mg/kg on the basis of cisplatin treatment once a day, and the amifostine group carries out intraperitoneal injection of amifostine 200mg/kg on the basis of cisplatin treatment once a day.

Starting with DAY 1, mice body weight and tumor volume were recorded daily, and euthanized material was taken after body weight and tumor volume were recorded on DAY 25. Blood was collected, and tumor tissue of the mice was rapidly collected, weighed, kidneys were collected, washed in physiological saline, observed and photographed. The right kidney was stripped of surface adipose tissue and mucosa, fixed to 4% paraformaldehyde for histopathological examination, and the remainder marked and placed in a-80 ℃ refrigerator for other relevant examination.

The kidney tissue was excised, total protein extracted, protein quantification was performed by BCA method, and expression of KIM-1, p53 and CLEAVED CASPASE in kidney tissue was detected by conventional Western Blotting.

Histopathological examination kidney tissue was fixed in 4% paraformaldehyde, embedded in normal paraffin, cut into 4 μm thick sections and stained with normal H & E. Observing, photographing and analyzing under the light microscope.

Renal function testing levels of creatinine (BUN) and urea nitrogen (CREA) were measured using a dry biochemical analyzer (Jiangsu Kang Shang biomedical technologies Co.).

Inflammatory response detection the levels of TNF- α in serum and IL-1β and IL-6 in the kidneys were detected using a commercial ELISA kit (Xinbo biosciences Co., ltd.).

Electrolyte disorder detection, namely detecting the level of electrolyte magnesium ions and calcium ions in serum by using a commercial kit (biological engineering Co., ltd.).

The results are shown in FIG. 10 to FIG. 11. Compared with the CON group, the tumor volume of the other groups is obviously reduced, and the combination of the positive drug amifostine or ORL and cisplatin does not influence the tumor volume, and does not weaken the anti-tumor activity of the cisplatin. During the whole experiment, one mouse died in the CIS group on day 16, one mouse died in the combination group of amifostine and cisplatin, which is a positive drug, on days 12 and 18, while none of the CON and ORL groups died. Renal function tests show that cisplatin induces obvious increases in BUN and CREA, that ORL and amifostine obviously reverse the effects of cisplatin, and that the reverse effects of ORL are similar to amifostine. Inflammatory factor level testing showed that ORL can dose-dependently reduce levels of TNF- α, IL-1β and IL-6. Electrolyte level detection showed that cisplatin significantly induced hypomagnesian, hypocalcemic, suggesting electrolyte turbulence, which was almost completely reversed by ORL.

H & E staining is also known as hematoxylin & eosin staining. After staining, the nuclei appeared bluish violet and the cytoplasm appeared pink. The H & E staining results of the kidneys show that compared with the CON group, the kidneys of the mice in the CIS group are obviously damaged, vacuoles or swells, and the pathological changes can be obviously improved after ORL and amifostine are dosed. Western Blotting results of kidney tissues show that the expression of a biomarker KIM-1 for renal injury induced by cisplatin is obviously increased, the expression of pro-apoptotic proteins p53 and CLEAVED CASPASE is increased, the changes are reversed to different degrees in each group of ORL treatment, and the reversing effect of ORL is obviously better than that of a positive drug amifostine. These results suggest that ORL, while improving cisplatin-induced kidney injury in tumor-bearing mice, does not affect the antitumor efficacy of cisplatin, and is better than the positive control drug amifostine in some indicators.

It is particularly noted that the ORL administered by intraperitoneal injection of this example did not find any kidney stones or calcium oxalate crystals in the kidneys. It has been reported that oral ORL competitively inhibits the binding of oxalic acid to calcium by inhibiting lipolysis, resulting in increased renal oxalic acid excretion. Finally, oxalic acid in the kidney tubules is supersaturated, and calcium oxalate crystals or kidney stones are formed. Thus, this suggests that the side effects of ORL-induced kidney stones or calcium oxalate crystallization may be related to the mode of administration. The ORL is administered by intraperitoneal injection in this example, which avoids inhibition of local lipolysis of the gastrointestinal tract, does not affect the combination of oxalic acid and calcium in the gastrointestinal tract, and does not promote increased absorption of oxalic acid by the gastrointestinal tract and increased excretion of renal oxalic acid. Thus, intravenous administration of ORL will avoid the potential renal injury effects of oral administration of ORL.

The pharmacological experiment results show that the ORL has remarkable effects of inhibiting the renal toxicity of cisplatin and protecting the kidney, does not influence the anti-tumor efficacy of cisplatin in vivo and in vitro, and has application prospect of developing medicaments for improving cisplatin-induced renal injury.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

Claims (10)

1. Use of orlistat or a pharmaceutically acceptable salt thereof in the preparation of a product for preventing and/or treating renal injury. 2. The use according to claim 1, characterized in that the renal injury is drug-induced renal injury; Preferably, the drug-induced renal injury is an anti-tumor drug; Preferably, the anti-tumor drug is a platinum anti-tumor drug; Preferably, the platinum anti-tumor drug is cisplatin. 3. The use according to claim 1, characterized in that the renal injury comprises renal injury induced by cisplatin. 4. The use according to claim 3, characterized in that the cisplatin-induced renal injury includes acute renal injury, chronic renal injury and electrolyte disorders related thereto. 5. The use according to claim 4 is characterized in that the product prevents and/or treats cisplatin-induced renal injury by improving and reversing electrolyte disorders induced by cisplatin; and or, the product prevents and/or treats cisplatin-induced renal injury by reducing the expression level of inflammatory factors; and or, the product prevents and/or treats cisplatin-induced renal injury by reducing creatinine and urea nitrogen. 6. The use according to claim 5, characterized in that the electrolyte disorder includes at least one of hypomagnesemia and hypocalcemia; and/or the inflammatory factors include TNF-α, IL-1β and IL-6. 7. Use of orlistat or a pharmaceutically acceptable salt thereof in the preparation of a drug for reducing cisplatin nephrotoxicity. 8. The use according to claim 7 is characterized in that the drug reduces the nephrotoxicity of cisplatin by inhibiting DNA damage and oxidative stress induced by cisplatin; and/or, the drug reduces the nephrotoxicity of cisplatin by inhibiting epithelial cell death and apoptosis induced by cisplatin; and/or, the drug reduces the nephrotoxicity of cisplatin by inhibiting endoplasmic reticulum stress of epithelial cells and activation of MAPK pathway induced by cisplatin. 9. The use according to any one of claims 1 to 8, characterized in that the pharmaceutically acceptable salt comprises at least one of hydrochloride, hydrobromide, sulfate, phosphate, acetate, citrate, lactate, ascorbate, maleate, tartrate, malate, gluconate, sulfonate, citrate, benzoate, benzenesulfonate, carbonate, methanesulfonate, stearate, nitrate, valerate or succinate. 10. A medicament comprising orlistat or a pharmaceutically acceptable salt thereof and cisplatin; Preferably, the drug further comprises a pharmaceutically acceptable excipient.