CN111393278B

CN111393278B - Usnea longissima derivative and application thereof in preparation of medicine for treating gallbladder cancer

Info

Publication number: CN111393278B
Application number: CN202010309840.1A
Authority: CN
Inventors: 刘涛; 李燕军; 陈海云
Original assignee: Shanxi Bethune Hospital of Shanxi Academy Of Medical Sciences
Current assignee: Shanxi Bethune Hospital of Shanxi Academy Of Medical Sciences
Priority date: 2020-04-20
Filing date: 2020-04-20
Publication date: 2020-11-20
Anticipated expiration: 2040-04-20
Also published as: CN111393278A

Abstract

The invention provides a usnea derivative and application thereof in preparing a medicine for treating gallbladder cancer, wherein the usnea derivative has a chemical name of 3, 6-diacetyl-2, 7, 9-trihydroxy-8, 9 b-dimethyl-1 (9bH) -dibenzofuranone, has an effect of inhibiting the activity of human gallbladder cancer cells, and has small toxic effect on normal cells. The compounds can be used alone or in combination with other drugs in the treatment of gallbladder cancer. The inventor unexpectedly finds that the compound provided by the invention can achieve better effect of inhibiting gallbladder cancer cells and effectively relieve the side effect of cisplatin when being used together with the cisplatin. Is a medicine with great application potential for treating gallbladder cancer.

Description

Usnea longissima derivative and application thereof in preparation of medicine for treating gallbladder cancer

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a Usnea longissima derivative and application thereof, in particular to application of the Usnea longissima derivative in preparation of a medicine for treating gallbladder cancer.

Background

Gallbladder cancer is a rare malignant tumor, and accounts for the first part of gallbladder malignant tumors, and is the most common malignant tumor of the biliary tract system, wherein adenocarcinoma is the main tumor, and accounts for about 82%. The incidence rate of gallbladder cancer is rapidly increased in recent years, and the gallbladder cancer is located at the 5 th position in digestive tract tumors. Gallbladder cancer is considered to be one of the most aggressive and lethal malignancies. The gallbladder cancer of China is relatively high, and the gallbladder cancer becomes a malignant disease which seriously threatens the lives of the masses of people day by day. According to Global Cancer Statistics (2012), in developed regions, the age-normalized rate per 100,000 of gallbladder Cancer has an age-normalized incidence of 2.0 and an age-normalized mortality of 1.4; in less developed areas, age-normalized morbidity rates are 2.4 and age-normalized mortality rates are as high as 2.0, which means that the final outcome of patients is mostly death as long as the disease is developed. Gallbladder cancer with occult onset often coexists with benign gallbladder diseases, most often with gallbladder calculus and cholecystitis, and chronic stimulation of calculus is an important pathogenic factor. The early stage of the gallbladder cancer has no specific clinical symptoms and physical signs, the clinical manifestations of the gallbladder cancer are often covered by symptoms of other diseases such as gallbladder calculus and the like, most patients have a medium-late stage diagnosis, the chance of radical excision is low, the prognosis is poor, and the 5-year survival rate after operation is less than 5%.

At present, the pathogenesis of gallbladder cancer is not clear, and the epidemiological investigation results show that gallstones, gallbladder adenomas, hyperplasia of gallbladder adenomatous muscles, biliary tract bacterial infection bile acid metabolic disorder, gastrectomy, high fat diet, smoking, alcohol addiction and the like are all risk factors for the occurrence of the gallbladder cancer.

Due to the characteristics of high malignancy, easy early metastasis, difficult early detection, insensitivity to chemotherapeutic drugs and the like, most patients are diagnosed in middle and advanced stages. At present, the treatment methods of gallbladder cancer include surgery, chemotherapy, radiotherapy, interventional therapy, gene therapy and the like. The chemotherapy drugs for gallbladder cancer commonly used in clinic at present comprise mitomycin, fluorouracil, paclitaxel, cisplatin and the like, which all have advantages and disadvantages, for example, mitomycin has high effective rate, but bone marrow suppression effect is also strong, and cisplatin has the advantages of wide anticancer spectrum and strong effect, but is easy to cause renal toxicity. In view of the above situation, it is of great clinical significance to find a drug or a pharmaceutical composition having a therapeutic effect on gallbladder cancer.

The Changsuo is Usnea longissima Ach of Usnea of Usneaceae, also named Yunwei, Laodianxu, devil's rush, Tianfluffcao, Tianshacao, Centipeda fragrans, Centipeda vittata, Osmanthus fragrans, Gloiopeltis furcata, Xuefeng vine, mountain fine dried noodles and Qifa all in a linear shape and is distributed in Daxingan mountain, Changbai mountain, Xinjiang, Tibet, Hubei, Anhui, Zhejiang river and other places in China. Various known derivatives, such as substituted mono-benzene, depside, anthraquinone, dibenzofuran and the like, have biological activities of resisting bacteria, diminishing inflammation, resisting oxidation, resisting tumors and the like. Is the research hotspot of the current natural products. Various compounds have been isolated and identified from Usnea longissima, such as ethyl gibberellic acid, friedelin, beta-coumarol, beta-sitosterol, methyl 2, 4-dihydroxy-3, 6-dimethylbenzoate, damasconic acid, zeugo terpene, ethyl enterolacate, 3 beta-hydroxy-mucor-5-ene, oleanolic acid, usnic acid, methyl enterolacate, and 4-methyl-2, 6-dihydroxybenzaldehyde. The inventor unexpectedly discovers that one of the usnea longissima and the derivatives thereof has the function of inhibiting the activity of gallbladder cancer cells, and is expected to be applied to clinic as a medicine for treating gallbladder cancer.

Disclosure of Invention

The invention aims to provide a compound with the activity of inhibiting gallbladder cancer cells and a derivative thereof, wherein the compound has obvious inhibition effect on the gallbladder cancer cells and small cytotoxicity.

In a first aspect, the present invention provides a compound of formula I, and pharmaceutically acceptable salts thereof,

wherein R is₁And R₂Independently selected from H, -OH, -CN, C_1-6Straight chain/branched alkyl, -OC_1-6Alkyl, -N (C)_0-10Alkyl) (C_0-10Alkyl), -CO (C)_0-10Alkyl), -CON (C)_0-10Alkyl) (C_0-10Alkyl groups).

Preferably, R₁And R₂Independently selected from H, -OH, -CH₃、-C₂H₅、-COCH₃。

Most preferably, R₁Is selected from-OH, -OCH₃，-OC₂H₅，R₂Selected from-COOH, -COOCH₃，-COOC₂H₅

In the most preferred embodiment of the present invention, said R₁is-OH, R₂Is selected from-COCH₃The structure of the compound is shown as the formula (II):

the chemical name of the compound is 3, 6-diacetyl-2, 7, 9-trihydroxy-8, 9 b-dimethyl-1 (9bH) -dibenzofuranone, which is extracted from a natural medicine, namely Usnea longissima, and the compound is named as Usnea longissima element.

The pharmaceutically acceptable salts of the present invention include: potassium, sodium or ammonium salts, or salts formed from methylamine, ethylamine, ethanolamine. The salt can be in the form of a mono-salt, di-salt, tri-salt. Preferred according to the invention are the mono-, di-and trisodium salts of the compounds of formula (II).

In a second aspect, the present invention provides a pharmaceutical composition comprising a compound represented by formula (I) or formula (ii), or a pharmaceutically acceptable salt thereof, as a pharmaceutically active ingredient.

Preferably, the pharmaceutical composition comprises a therapeutically effective amount of a compound of formula (ii). The pharmaceutical composition may be formulated into the following dosage forms: tablet, capsule, injection, powder for injection, suspension, and emulsion. The pharmaceutical preparation is preferably a unit preparation comprising an effective dose of the active compound, preferably varied or adjusted between 0.1mg and 10mg, in particular 0.1mg, 0.5mg, 1mg, 2mg, 4mg, 5mg, 10 mg.

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant or carrier, and the adjuvant or carrier is determined according to a specific administration method of the composition. In the practice of the present invention, the pharmaceutical composition may be administered by gastrointestinal (intravenous, intramuscular, intradermal, and subcutaneous routes), parenteral (oral, nasal, sublingual routes), intraperitoneal (i.p.) routes.

In a particular mode of administration of the invention, the composition is administered intravenously, and therefore, the pharmaceutical composition further comprises antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with blood. More preferably, the pharmaceutical composition can also comprise a suspending agent, a solubilizer, a thickening agent and a stabilizing agent.

In the context of the administration of the present invention, the dosage to a subject is determined by the potency of the particular active compound and the condition of the subject, as well as the body weight or body surface area of the subject to be treated, to produce a beneficial therapeutic response in the subject, and also by the presence, nature and extent of any adverse side effects associated with administration in the particular subject.

In certain embodiments, the pharmaceutical compositions may be used alone or in combination with other types of pharmaceutical formulations and/or methods of treatment.

Such other classes of pharmaceutical formulations and/or methods of treatment include, but are not limited to: immunosuppressants, targeted antineoplastic drugs, non-steroidal anti-inflammatory drugs, anti-tumor vaccines, EGFR tyrosine kinase inhibitors, adoptive cellular immunotherapy or radiotherapy.

Specifically, the drugs that can be used in combination with the pharmaceutical composition of the present invention include: one or more of mitomycin, fluorouracil, paclitaxel, cisplatin, carboplatin and oxaliplatin. In the invention, the inventor unexpectedly finds that the compound shown in the formula (I) or the formula (II) provided by the invention can achieve better effect of inhibiting gallbladder cancer cells and effectively relieve the side effect of cisplatin when being used together with cisplatin.

Cisplatin is currently a first-line medicine for treating various solid tumors and is also one of the common gallbladder cancer chemotherapeutics in the present clinical stage. Cisplatin belongs to a cell cycle non-specific drug, can inhibit the DNA replication process of cancer cells, damages the structure on the cancer cell membrane, and has broad-spectrum anticancer effect. Therefore, cisplatin has high cytotoxicity and great side effect in clinical application, and is particularly easy to cause renal toxicity.

In a third aspect, the present invention provides a pharmaceutical composition comprising a compound represented by formula (I) or formula (ii) or a pharmaceutically acceptable salt thereof as a first active pharmaceutical ingredient; the pharmaceutical composition also comprises cisplatin as a second active pharmaceutical ingredient.

The mass ratio of the first active pharmaceutical ingredient to the second active pharmaceutical ingredient in the compound pharmaceutical composition is 1: 1-10; more preferably, the mass ratio of the first active pharmaceutical ingredient to the second active pharmaceutical ingredient is 1: 1-3.

The second active pharmaceutical ingredient is one or the combination of more than two of mitomycin, fluorouracil, paclitaxel, cisplatin, carboplatin and oxaliplatin, preferably cisplatin.

Preferably, the compound pharmaceutical composition further comprises pharmaceutically acceptable adjuvants or carriers, and the selection of the specific adjuvants or carriers is as described above.

The active pharmaceutical ingredients in the compound pharmaceutical composition can form a pharmaceutical preparation with pharmaceutically acceptable auxiliary materials or carriers. Preferably, the compound pharmaceutical composition is in the form of sterile powder, injection solution or suspension. The term "pharmaceutically acceptable carrier" refers to one or more compatible solids, liquids, diluents, and the like, suitable for administration to the human body, which carrier does not destroy the therapeutic effect of the active compound.

The administration mode and effective dose of the compound pharmaceutical composition are determined according to the specific stage of the disease to be treated, the age and physical condition of the patient and the duration of the disease, and can be selected according to the actual needs within the knowledge and time range of the skilled person. In general, an effective amount is one that is sufficient to cause a favorable phenotypic change, such as alleviation or elimination of symptoms, permanent arrest of progression of the disease, or delay of onset of the disease, etc.

In a fourth aspect, the invention provides an application of a compound shown in formula (I) or (II) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition which comprises the compound shown in formula (I) or (II) or the pharmaceutically acceptable salt thereof as an active ingredient in preparing a medicament for treating gallbladder cancer.

Term C in the present invention_0-10Alkyl radical, C₀Alkyl means H, thus, C_0-10Alkyl includes H, C₁Alkyl radical, C₂Alkyl radical, C₃Alkyl radical, C₄Alkyl radical, C₅Alkyl radical, C₆Alkyl radical, C₇Alkyl radical, C₈Alkyl radical, C₉Alkyl radical, C₁₀An alkyl group.

The term "unit formulation" as used herein refers to the smallest unit package of each pharmaceutical preparation containing an appropriate amount of active compound, e.g., a unit formulation in a capsule refers to a capsule, a unit formulation in a tablet refers to a tablet, a unit formulation in an injection refers to an injection, other types of formulations, and so forth.

The compound shown in the formula (I) or (II) and the pharmaceutically acceptable salt thereof have the following advantages when being used as a medicine for treating gallbladder cancer:

(1) the compound shown in the formula (I) or (II) has obvious inhibition effect on human gallbladder cancer cells as an active ingredient, which indicates that the compound has the potential of clinical application;

(2) compared with chemotherapy drugs commonly used in clinic, the compound shown in the formula (I) or (II) has lower cytotoxicity and stronger safety, and the drug has smaller side effect in a mouse test;

(3) in the application, the compound shown in the formula (I) or (II) and cisplatin are used together, so that the synergistic effect is achieved, and the side effect caused by the cisplatin can be obviously reduced.

Drawings

FIG. 1 shows the inhibitory effect of usnin and cisplatin on human gallbladder cancer cells.

FIG. 2 shows the inhibitory effect of longiusnine and fluorouracil, which are compounds represented by formula (II), on human gallbladder cancer cells.

FIG. 3 shows the cytotoxic effect of usnin and cisplatin on macrophages for the compounds represented by formula (II).

FIG. 4 shows the cytotoxic effect of longiusnine and fluorouracil, which are compounds represented by formula (II), on macrophages.

FIG. 5 is a graph showing the change in body weight of nude mice under the action of drugs.

FIG. 6 survival curves of nude mice under drug action.

FIG. 7 tumor weight distribution under drug action.

FIG. 8 is a graph showing the tumor volume distribution of nude mice under the action of drugs.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 preparation of usnin injection

S1: preparing a solution, namely dissolving 10mg of the compound usnic element shown in the formula II in 20mL of sodium chloride injection to prepare a liquid medicine with the medicine concentration of 0.5 mg/mL;

s2: activated carbon treatment, namely adding activated carbon for needles, the amount of which is 0.2 percent of the total amount of the solution, into the liquid medicine for adsorption treatment for 0.5 hour;

s3: primarily filtering, namely filtering the liquid medicine by using filter paper;

s4: filtering to remove bacteria, and removing bacteria and heat source with microporous membrane with pore diameter of 0.22 μm;

s5: subpackaging the liquid medicine by taking 2mL as a unit, and encapsulating to obtain the injection.

EXAMPLE 2 preparation of Usnesin-cisplatin Compound injection

S1: preparing a solution, namely dissolving 10mg of the compound usnine shown in the formula II and 30mg of cis-platinum together in 20mL of sodium chloride injection to prepare a compound liquid medicine with 0.5mg/mL of usnine and 1.5mg/mL of cis-platinum;

The applicant needs to emphasize that the ratio of the usnin to the cisplatin can be any ratio between 1:1 and 10, the specific examples of the application only show the ratio of 1:3, and the skilled person can also prepare compound liquid medicines with the ratio of the usnin to the cisplatin being 1:1, 1:2, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1: 10.

EXAMPLE 1 Usnenin has inhibitory effect on gallbladder cancer cells

All experimental GBC-SD cell lines used herein were human gallbladder cancer cells, purchased from ATCC (American Type Culture Collection, Manassas, Va.). Culture conditions of GBC-SD cell line: the culture medium 1640 containing 10% fetal calf serum, 100U/mL penicillin and 100. mu.g/mL streptomycin was used at 37 ℃ and 5% CO₂The culture was carried out under the conditions of (1), and cells in logarithmic growth phase were taken in the specific experimental operation.

GBC-SD cell processing step: taking out the human gallbladder cancer cells frozen in liquid nitrogen, immediately putting the human gallbladder cancer cells into a water bath at 37 ℃ for resuscitation for 10 minutes; transferring the recovered cell suspension into a clean centrifuge tube, adding 10mL of serum-free 1640 culture medium, gently mixing uniformly, centrifuging at 4000rpm for 5 minutes, then removing supernatant, and repeating for three times; adding 1mL of 10% FBS-1640 culture medium, uniformly mixing, placing in a cell culture box at 37 ℃, and culturing under the condition of 5% carbon dioxide; fresh medium was replaced every 2 days.

Purpose of the experiment: cisplatin and fluorouracil are used as controls to detect whether the longiusnine has obvious inhibition effect on GBC-SD human gallbladder cancer cells.

The experimental steps are as follows:

s1: gently washed twice with PBS buffer, then cell counted and plated evenly into 96-well plates at a cell density of 8000 cells/well, 200. mu.L per well volume, at 37 ℃ with 5% CO₂And culturing in an incubator with saturated humidity for 24h, designing the positions and the number of the control holes and the zero adjusting holes, and adding 200 mu L of sterile PBS into each hole on the periphery of a 96-hole plate in one circle in order to avoid influencing the experimental result after the concentration is changed due to water evaporation;

s2: the usnin is prepared into four liquid medicines with four gradient concentrations of 0.5 mu M, 1.0 mu M, 2.0 mu M and 5.0 mu M by taking sodium chloride injection as a solvent, in addition, the contrast medicines of cisplatin and fluorouracil are also prepared into four liquid medicines with four gradient concentrations of 0.5 mu M, 1.0 mu M, 2.0 mu M and 5.0 mu M by taking the sodium chloride injection as a solvent, and the two combinations of the contrast medicine and the experimental medicine of the experiment are respectively carried out in two 96-well plates simultaneously;

s3: after the cells are cultured for 24 hours, sucking out the old culture medium of each hole by using a pipette gun, respectively adding the experimental medicine and the control medicine with corresponding concentrations, adding the same amount of sodium chloride injection into the control hole and the zero setting hole, and continuously culturing for 24 hours.

S4: coloring, carefully weighing MTT, preparing a solution with the concentration of 5mg/mL by using sterile PBS, taking out a 96-well plate after the medicine acts for 24 hours, adding 20 mu L of MTT solution into each well, incubating for 4 hours in an incubator to avoid sucking away cells, sucking out old culture medium by using a sterile syringe, adding 150 mu L of DMSO into each well, and incubating for 10 min.

S5: and (3) carrying out color comparison, selecting 490nm wavelength, measuring the light absorption value of each pore on an enzyme-linked immunosorbent detector, and recording the result.

Statistical analysis: each group of data in the experiment is instrument detection data, statistical analysis software SPSS 17.0 is used, single-factor analysis of variance and t test are adopted for statistical analysis, and P is less than 0.05 to indicate that the difference has statistical significance.

The experimental results are as follows: FIG. 1 is a graph showing the effect of usnin and cisplatin on the growth of gallbladder cancer cells, and FIG. 2 is a graph showing the effect of usnin and fluorouracil on the growth of gallbladder cancer cells. As shown in figures 1 and 2, the proliferation and growth of each cell strain are reduced along with the increase of the drug concentration, and the inhibition effect of the longiusnin on the cancer cells of the gall bladder is obviously different from that of the control group when the concentration of the longiusnin is 2.0 mu M compared with the inhibition tendency of the cis-platinum and the fluorouracil on the cancer cells of the gall bladder in figures 1 and 2. Although the inhibition effect of the experimental drug on the gallbladder cancer cells is not significantly different compared with the control drug at other concentrations (0.5, 1.0 and 5.0 mu M), it can be seen from the figure that the inhibition effect of the usnea longissin on the gallbladder cancer cells and the inhibition effect of cisplatin and fluorouracil are stronger at the drug concentration of 2.0-5.0 mu M.

Effect example 2 toxic Effect of usnin on Normal cells

This experiment utilized RAW264 macrophages to detect the toxicity of the active compound usnin described herein to normal cells. RAW264 macrophages were purchased from ATCC. RAW264 cells were cultured in DMEM low-glucose medium containing 10% Fetal Bovine Serum (FBS). Freezing tube for freezing cells from liquid nitrogenTaking out from the tank, rapidly placing into a 37 ℃ water bath tank, shaking to dissolve rapidly, transferring the dissolved cell sap into a centrifuge tube with fresh culture medium added in advance to avoid influence on cell growth by DMSO added during cell freezing, placing into a centrifuge at 1000rpm/min for centrifuging for 5min, taking out, discarding supernatant, transferring the cells into a culture bottle, adding fresh culture medium, shaking uniformly at 37 ℃, and containing 5% CO₂The incubator of (4) is used for conventional culture.

The growth of the cells is observed under a microscope, and when the cells are relatively dense under the microscope, the cells are ready for passage. In a sterile operating platform, pouring the culture solution in the culture bottle, quickly adding sterile PBS for washing once, then adding 0.25% trypsin (containing 0.02% EDTA) digestive juice, incubating for 5min, adding a fresh culture medium, blowing with a rubber-tipped dropper, blowing off the cells on the bottle wall, adding a part of the cells into a new culture bottle for continuous culture, and carrying out the experiment on the remained cells.

Purpose of the experiment: cisplatin and fluorouracil were used as controls to test the toxic effects of usnin on RAW264 macrophages.

The experimental steps are as follows:

s1: the cells were seeded and the positions and number of control and zero wells were designed so that 200. mu.L of sterile PBS per well was added around the periphery of the 96-well plate in order to avoid affecting the results of the experiment after concentration changes due to water evaporation. Dispersing macrophage in DMEM culture medium to obtain single cell suspension, counting cells under microscope, inoculating macrophage to 96-well plate with about 5000 cells per well, 200 μ L per well, placing at 37 deg.C and 5% CO₂And culturing for 24h in an incubator with saturated humidity;

s2: the usnin is prepared into four liquid medicines with four gradient concentrations of 1.0 mu M, 2.0 mu M, 5.0 mu M and 10.0 mu M by taking sodium chloride injection as a solvent, in addition, the contrast medicines of cisplatin and fluorouracil are also prepared into four liquid medicines with four gradient concentrations of 1.0 mu M, 2.0 mu M, 5.0 mu M and 10.0 mu M by taking the sodium chloride injection as a solvent, and the two combinations of the contrast medicine and the experimental medicine of the experiment are respectively carried out in two 96-well plates simultaneously; s3 drug action, S4 color generation and S5 colorimetric operation are the same as those in example 1.

The experimental results are as follows: FIGS. 3 and 4 are graphs showing the cytotoxic effects of usnin and cisplatin, usnin and fluorouracil on RAW264 macrophages, respectively. As shown in fig. 3 and fig. 4, compared with cisplatin and fluorouracil, the toxicity of usnin to macrophages is significantly reduced, and the effect of reducing toxicity is more obvious when the drug concentration is higher, which is sufficient to indicate that usnin has lower cytotoxicity, better safety and great clinical application potential.

EXAMPLE 3 Usnesin has inhibitory effect on tumor in nude mice bearing tumor

Purpose of the experiment: cisplatin and fluorouracil are used as controls to detect the inhibition effect of the usnin on tumor-bearing nude mice tumors, and simultaneously detect the inhibition effect of the compound injection of the usnin and cisplatin on tumor-bearing nude mice tumors.

The experimental steps are as follows:

s1: establishing a human gallbladder cancer nude mouse transplantation tumor model: the experimental animals are 50 SPF female nude mice, the mice age is 7-8 weeks, and the weight is 18-20 g. Taking GBC-SD cells in logarithmic growth phase, and preparing into 1 × 10 with sodium chloride injection⁶Nude mice were injected subcutaneously at a concentration of 0.5 mL/mouse in total cells and fed under normal conditions. When the tumor volume reaches about 200mm after 2-3 weeks³When the model is established, the model building is completed.

S2: nude mice were randomly divided into five groups, and all the nude mice were administered tail vein, wherein the longisunitin injection (specification 1mg/2mL, 100-fold dilution, 600. mu.L) prepared in example 1 was administered to experiment 1 group, the longisunitin-cisplatin compound injection (specification 1mg:3mg/2mL, 100-fold dilution, 600. mu.L) prepared in example 2 was administered to experiment 2 group, the commercially available cisplatin injection (specification 10mg/2mL, 100-fold dilution, 600. mu.L) was administered to control 1 group, the fluorouracil injection (specification 0.25g/10mL, 100-fold dilution, 600. mu.L) was administered to control 2 group, and 600. mu.L of sodium chloride injection was administered to the tail vein of nude mice in blank control group. The treatment frequency is 2 times per week, the nude mice are treated on Monday and Wednesday respectively, the nude mice are fed normally in the rest time, the survival rates of five groups of nude mice are counted per week, the weight change of the nude mice is measured per week, the nude mice are observed for activity state, the survival nude mice are killed after being kept for six weeks, and the weight and the volume of the tumor are measured after the tumor is stripped.

The experimental results are as follows:

weight: as shown in fig. 5, the weight of mice treated by the usnin and the compound preparation shows a slow rising trend, which indicates that the growth of tumor is effectively inhibited, while the weight of nude mice treated by cisplatin and fluorouracil is obviously reduced in the later period, and the nude mice can be observed to be increasingly leaner and leaner along with the longer and bigger tumor in the visual sense, especially, the nude mice treated by cisplatin have higher death rate and the worst overall state. It can be seen that the weight loss of nude mice is obviously slowed down after the usnic is combined on the basis of cisplatin, which indicates that the usnic can effectively relieve the side effect generated by cisplatin treatment.

Survival rate: as shown in fig. 6, in all the drug treatment groups, the survival rate of the nude mice treated with usnin was the highest after 6 weeks, and reached 100%, and the survival rate of the nude mice treated with usnin-cisplatin compound preparation was 80% after 2 deaths after 6 weeks. The highest mortality rate is in the cisplatin treatment group, the mortality rate of the nude mice is up to 60%, the number of the nude mice after 6 weeks of treatment is equivalent to that of the blank control group, and the state of the remaining alive nude mice after 6 weeks of treatment is also very poor. As can be seen from the survival rate curve, compared with the existing first-line clinical drugs of cisplatin and fluorouracil, the usnea longissin and the compound preparation containing the usnea longissin provided by the application have higher safety and have the potential of being further applied to clinic.

And (4) adverse reaction observation: the drug treatment group mice do not show the performance of reduced activity, listlessness and the like, the feeding of the mice is not obviously reduced, the mice do not have obvious emaciation and death during the experiment, and the skin of the transplanted tumor is not broken or eroded.

Tumor volume and weight: removing the liquefied tumors, and then counting the weight and volume distribution of each group of tumors, as shown in fig. 7 and 8, wherein the volume and weight distribution trend of each group of tumors are basically consistent, the weight and volume of the tumors of the nude mice of the blank control group are the largest, the tumor growth of the nude mice of the cisplatin and fluorouracil treatment group is inhibited, and the weight and volume of the tumors are relatively reduced, but the relatively smallest weight and volume of the tumors are the usnea longissin and usnea longissin-cisplatin compound injection, especially the weight and volume of the tumors of the compound injection treatment group are the smallest, which indicates that the tumor inhibition effect of the compound injection is the best. The tumor liquefaction phenomenon of the cisplatin and fluorouracil treatment group is obvious, and the probability of tumor liquefaction is about 30-40%; unexpectedly, it was found that the tumors of nude mice in the treatment group of usnea longissima were substantially free from liquefaction. Meanwhile, the sizes and the volume distributions of the tumors of the live nude mice in the cisplatin and fluorouracil treatment groups are relatively discrete, which shows that the cisplatin and fluorouracil have unobvious treatment effects on certain individuals.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of a usnea derivative or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the usnea derivative or the pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating gallbladder cancer, wherein the usnea derivative has a chemical name of 3, 6-diacetyl-2, 7, 9-trihydroxy-8, 9 b-dimethyl-1 (9bH) -dibenzofuranone, and the structural formula is shown as the following formula (II):

(II)。

2. use according to claim 1, wherein the pharmaceutically acceptable salt is selected from the potassium, sodium or ammonium salt, in the form of a mono-, di-or tri-salt.

3. The use of claim 1, wherein the usnea longissima derivative represented by formula (II) or a pharmaceutically acceptable salt thereof is used as a pharmaceutically active ingredient in the pharmaceutical composition, and the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant and/or carrier.

4. The use of claim 3, wherein the adjuvants include antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood.

5. The use of claim 1, wherein the pharmaceutical composition comprises a unit preparation of a therapeutically effective amount of the usnea longissima derivative of formula (II) or a pharmaceutically acceptable salt thereof, wherein the dosage of the pharmaceutically active ingredient in the unit preparation is 0.1mg-10 mg.

6. The use of claim 1, wherein the pharmaceutical composition is used in combination with other types of pharmaceutical agents including immunosuppressants, targeted antineoplastic agents, non-steroidal anti-inflammatory drugs, anti-tumor vaccines or EGFR tyrosine kinase inhibitors.

7. The use according to claim 6, wherein the pharmaceutical formulation used in combination with the pharmaceutical composition is selected from one or a combination of two or more of mitomycin, fluorouracil, paclitaxel, cisplatin, carboplatin, oxaliplatin.

8. The use of claim 1, wherein the pharmaceutical composition comprises an usnea derivative represented by formula (II) or a pharmaceutically acceptable salt thereof as a first active pharmaceutical ingredient; the pharmaceutical composition also comprises a second active pharmaceutical ingredient, the mass ratio of the first active pharmaceutical ingredient to the second active pharmaceutical ingredient is 1:1-10, and the second active pharmaceutical ingredient is cisplatin.

9. The use of claim 8, wherein the mass ratio of the first active pharmaceutical ingredient to the second active pharmaceutical ingredient is 1: 1-3.