Application of amlodipine in preparation of medicine for treating echinococcosis

Abstract

The invention discloses an application of amlodipine in preparation of a medicine for treating echinococcosis. The amlodipine further comprises a pharmaceutically acceptable salt of the amlodipine and a pharmaceutical composition containing pharmaceutically acceptable auxiliary materials. An echinococcosis killing experiment result shows that when the highest action concentration of albendazole in an experiment is 80 [mu] M, the fatality rate of albendazole to protoscolex is only 2.5% + / -0.5%, when the action concentration of amlodipine is 20 [mu] M, the fatality rate of albendazole to protoscolex can reach 2.5% + / -0.5%, and when the action concentration of amlodipine is 60-80 [mu] M, the fatality rate of albendazole to protoscolex can reach 100%; microscopic experiments show that when the action concentration of amlodipine is 60 [mu] M, protoscolex can be killed only in 8 hours. Therefore, compared with albendazole for clinical adjuvant treatment of the echinococcosis, the amlodipine disclosed by the invention has the advantages of more obvious effect of killing protoscolex in vitro, less dosage, higher safety and quicker action, and has important reference value for developing a new drug for treating the echinococcosis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of development and application of anti-parasitic drugs, and relates to application of amlodipine, in particular to application of amlodipine in preparation of a drug for treating echinococcosis. BACKGROUND

[0002] Echinococcosis is a zoonosis caused by the larvae of Echinococcus granulosus, which parasitizes the liver, lungs and other important organs of intermediate hosts. The larvae of Echinococcus multilocularis can cause alveolar echinococcosis in intermediate hosts.

[0003] The intermediate hosts of alveolar echinococcosis are mainly humans and rodents. The cysts are different from those of cystic echinococcosis. The cysts grow in a infiltrative manner in the parasitized organs and form a grape-like cluster. This tumor-like proliferation of cysts severely damages the function of the organs. The 10-year mortality rate of patients after infection without treatment can be as high as 94%.

[0004] Currently, the treatment of echinococcosis is surgical removal of cysts, supplemented by benzimidazole drugs (albendazole and mebendazole). However, the effect of benzimidazole drugs in killing echinococcosis is not good, and long-term use can cause adverse reactions. In addition, benzimidazole drugs are broad-spectrum anti-parasitic drugs that mainly inhibit the polymerization and formation of microtubules of parasites by binding to microtubulin, thereby achieving the purpose of anti-parasitism. At the same time, the function of microtubulin in the host is also inhibited, and long-term use may cause irreversible damage to the host. Therefore, it is urgent to explore the value of drugs approved by PDA for the treatment of other diseases in the treatment of echinococcosis to replace benzimidazole drugs and provide new drug options for the treatment of echinococcosis. SUMMARY

[0005] The purpose of the present application is to provide the application of amlodipine in the preparation of a drug for treating echinococcosis, which also includes pharmaceutically acceptable salts thereof.

[0006] In the present application, amlodipine is a widely used dihydropyridine calcium channel blocker in clinical practice, mainly used for the treatment of hypertension and angina pectoris. Its mechanism of action is to selectively inhibit the entry of calcium ions across the membrane into vascular smooth muscle and myocardial cells, causing peripheral arterial vasodilation, thereby reducing blood pressure and reducing myocardial oxygen consumption. So far, there have been no reports of amlodipine being used to treat echinococcosis. In the screening of anti-echinococcosis drugs, the applicant found that amlodipine has an effective killing effect on echinococcosis. Its structural formula is as follows:

[0007]

[0008] In the present application, the echinococcosis is alveolar echinococcosis, and the echinococcus is multilocular echinococcus.

[0009] Further, the echinococcus includes at least one of its cyst, germinal layer, ascus, daughter cyst, cyst fluid or protoscolex.

[0010] The present application also provides a pharmaceutical composition for treating echinococcosis, which includes amlodipine, pharmaceutically acceptable salt thereof, and pharmaceutically acceptable adjuvant.

[0011] Further, the dosage form of the composition is tablet, granule, powder, capsule, oral liquid, oral agent, injection, implant or patch.

[0012] The present application further provides a method for killing echinococcus in vitro, which is to dissolve amlodipine or pharmaceutically acceptable salt thereof in a solvent, prepare a mother liquor, dilute it with the same solvent to obtain a dilute liquor, and then add it into a culture system of echinococcus.

[0013] Further, the solvent is DMSO, and the concentration of the dilute liquor is 10-80 μM; preferably 20-80 μM.

[0014] The present application has the following beneficial effects:

[0015] The results of the method for killing multilocular echinococcus in the present application show that, when the highest action concentration of albendazole in the experiment is 80 μM, the mortality rate of protoscolex is only 2.5%±0.5%, while when the action concentration of amlodipine in the present application is 20 μM, the mortality rate of protoscolex can reach 2.5%±0.5%, and when the action concentration of amlodipine is 60-80 μM, the mortality rate of protoscolex can be as high as 100%; the experiment of microscopic structural changes of ceritinib killing multilocular echinococcus shows that, when the action concentration of ceritinib is 60 μM, protoscolex can be completely killed in only 8 hours. Therefore, compared with albendazole in the art which is used for auxiliary treatment of echinococcosis in clinic, amlodipine in the present application has more significant effect on killing protoscolex in vitro, uses less amount, has higher safety, and has more rapid effect on killing echinococcus, which has important reference value for developing new drugs for treating echinococcosis. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The optical microscope (40x) pictures of the multilocular protoscoleces after being treated with 60 μM of amlodipine, albendazole and equal volume of DMSO in Example 1 for 24 hours and the pictures of the blank control without any treatment are shown in the following table. The first row of pictures are the pictures of the non-stained protoscoleces, and the second row of pictures are the pictures of the FDA (fluorescein diacetate) and PI (propidium iodide) staining pictures. D22 in the following pictures means amlodipine, ABZ means albendazole, and DMSO means dimethyl sulfoxide.

[0017] Figure 2 Statistical chart of the killing effect of amlodipine on the hydatid cysts in Example 1 using 60 μM of amlodipine, albendazole and an equal volume of DMSO for 24 hours in vitro;

[0018] Figure 3 Photographs of the morphological changes of the hydatid cysts in Example 2 using 60 μM of amlodipine and an equal volume of DMSO for 8 hours in vitro (magnification: 50x, 100x, 200x and 400x, respectively);

[0019] Figure 4 Photographs of the hydatid cysts in Example 3 using 10-80 μM of amlodipine and an equal volume of DMSO for 24 hours in vitro (microscope bright field and red-green fluorescence); D22 FDA+PI: photographs of the hydatid cysts after the combination of FDA and PI after the action of amlodipine; DMSO FDA+PI: photographs of the hydatid cysts after the combination of FDA and PI after the action of DMSO;

[0020] Figure 5 EC50 of amlodipine on the hydatid cysts in Example 3 50 Concentration statistical results. DETAILED DESCRIPTION

[0021] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0022] The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[0023] Example 1

[0024] This example provides a method for killing the hydatid cysts by amlodipine, and the specific experimental operation steps are as follows:

[0025] 1. Materials and methods

[0026] 1.1 Mouse model of Echinococcus multilocularis infection: Echinococcus multilocularis was obtained from the abdominal cavity of BALB / c mice preserved in the laboratory of the applicant. The protoscoleces were washed in PBS buffer containing 1% penicillin-streptomycin until the solution containing the protoscoleces was clear. The protoscoleces were then thoroughly resuspended using a Pasteur pipette, and 10 μL of the resuspended protoscoleces was dropped onto a glass slide (3 drops were taken as biological replicates). The number of protoscoleces in 3 drops of 10 μL was counted under a light microscope, and the average number was calculated. The number of protoscoleces in the resuspended solution was adjusted to 10 protoscoleces per μL. A 200-μL aliquot of the resuspended protoscoleces (approximately 2000 protoscoleces) was injected intraperitoneally into BALB / c mice (specifically, 6-8-week-old BALB / c mice purchased from the Experimental Animal Center of the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, weighing approximately 20 g). This completed the construction of the mouse model of Echinococcus multilocularis infection. Two months after infection, the Echinococcus multilocularis cystic tissue and protoscoleces were obtained from the abdominal cavity for efficacy evaluation.

[0027] 1.2 Protoscoleces collection and in vitro drug action: DMEM medium, Pasteur pipettes, culture dishes, 50-mL centrifuge tubes, glass slides, alcohol cotton, 75% alcohol, autoclaved surgical instruments, PBS buffer, and a sieve-equipped beaker were prepared. After the BALB / c mice were sacrificed by cervical dislocation, the body surface was disinfected by immersion in disinfectant alcohol. The abdominal cavity was then opened, and the Echinococcus multilocularis cystic tissue was removed onto the sieve-equipped beaker. The cystic tissue was then cut into pieces using scissors, and the protoscoleces in the broken cystic tissue were flushed into the beaker using PBS buffer. This process was repeated several times to ensure that the protoscoleces were fully flushed out. The sieve was removed from the beaker, and the beaker was left to stand for 5 minutes. The supernatant was then discarded, and the protoscoleces that had settled were collected in a 6-cm culture dish. PBS buffer was added to the dish, and the protoscoleces were evenly dispersed by blowing. The dish was left to stand for 5 minutes, and then the protoscoleces were rapidly gathered towards the center of the dish by rotating the dish clockwise. The surrounding liquid was then discarded, and PBS buffer was added to the dish to disperse the protoscoleces. The dish was left to stand for several minutes, and then the above process was repeated 4-5 times until the liquid was clear. The protoscoleces in the center of the dish were collected in a 50-mL centrifuge tube, and only 10 mL of the liquid was retained after standing. The protoscoleces in the centrifuge tube were mixed by blowing, and 3 drops of 10 μL of the liquid were collected on a glass slide. The number of protoscoleces was counted under a microscope, and the average number was calculated. Finally, the amount of liquid was adjusted so that there were 100 protoscoleces per 10 μL of liquid. In each well of a 96-well cell culture plate, 190 μL of DMEM medium was added, and then 10 μL of the protoscoleces mixture was added to each well after mixing. The culture was left to stand overnight. Eight concentrations of amlodipine (amlodipine was dissolved in dimethyl sulfoxide) were set up: 80 μM, 60 μM, 40 μM, 35 μM, 30 μM, 25 μM, 20 μM, and 10 μM, with 3 biological replicates for each concentration. The corresponding volume of DMSO was set up as a control, and a blank control (without drug treatment) was also set up. Albenza was set up as a positive control.

[0028] 1.3 Killing effect observation and staining: The oncospheres in the culture plate were washed with PBS buffer for 3 times, the morphology and activity of the oncospheres were observed under a microscope, 1 minute per well to determine whether the oncospheres were active, whether the oncospheres were disintegrated, and the killing effect of the drug on the oncospheres was preliminarily judged; then the FDA dye was diluted with Hanks buffer at a ratio of 1:4000, added to the culture well, and incubated at 37°C for 10 minutes. After 10 minutes, the FDA dye in the culture well was aspirated and discarded, the oncospheres in the culture well were washed with Hanks buffer for 3 times, 2 μL of PI dye was added, and the oncospheres were incubated at 2-8°C for 5 minutes. After aspirating and discarding the PI dye in the culture well, the oncospheres were resuspended with Hanks buffer, and photographed under a fluorescence microscope. If the oncospheres died, the oncospheres would emit red fluorescence after PI staining. If the oncospheres did not die, the oncospheres would emit green fluorescence after FDA staining. Note that the whole process should be operated in the dark.

[0029] 1.4 Drug killing effect statistics: The mortality rate of oncospheres was calculated based on the microscopic results, and the mortality rate (%) = (number of dead oncospheres in the drug group - number of dead oncospheres in the control group) / (total number of oncospheres in the culture well - number of dead oncospheres in the control group) x 100%.

[0030] 1.5 Difference statistical analysis: The data were analyzed by GraphPad Prism 9.5, the mortality rate of oncospheres was represented by mean ± standard deviation (SD), and then one-way ANOVA was selected for significant difference analysis. Note: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0031] 1.6 Reagents: The compounds amlodipine, albendazole and DMSO used in this experiment were purchased from MCE company.

[0032] 2. Results

[0033] The killing effect of amlodipine and albendazole on multilocular oncospheres is shown in Figure 1 , Figure 2 and Table 1.

[0034] Table 1 Killing rate of different concentrations of drugs on in vitro action of multilocular hydatid cysts

[0035]

[0036] From Figure 1 and Figure 2The results show that after 24 hours of action of 60 μM amlodipine on multilocular hydatid, the structure of the protoscolex is destroyed and no movement is observed under microscope, indicating that the drug has a lethal effect, and all the protoscoleces are colored by PI and are not colored by FDA; the protoscolex structure of the albendazole and DMSO control groups is intact, and most of the protoscoleces can be observed to have movement under microscope, and the activity is not obviously affected and almost no protoscolex is colored by PI, indicating that the lethal effect of albendazole on protoscolex is not obvious. The results in Table 1 show that when the concentration of amlodipine is 60-80 μM, the lethal rate of protoscolex is 100%, and when the concentration of the drug is as low as 35 μM, the lethal rate of protoscolex is 66.7%±3.4%, showing an obvious killing effect on protoscolex and the effect is obviously better than that of albendazole; for albendazole, the overall effect of killing protoscolex is poor, and the change of the concentration of the drug does not obviously change the killing effect on protoscolex.

[0037] Example 2

[0038] This example provides the microscopic structural changes of amlodipine in killing multilocular hydatid, and the specific experimental operation steps are as follows:

[0039] 1. Materials and methods

[0040] The BALB / c mice infected with multilocular hydatid according to the modeling in Example 1 of the present application are treated by breaking the neck, and the body surface is disinfected, the peritoneal cavity is cut open, and the hydatid cyst tissue is taken out to a beaker bound with a sieve, the cyst tissue is cut and then washed with PBS buffer containing 1% penicillin-streptomycin until the protoscolex is washed out into the beaker, then the protoscolex in the beaker is repeatedly washed according to the above method, the washed protoscolex is transferred to a 50 ml centrifuge tube for counting, then the protoscolex is cultured in a 96-well cell culture plate, 100 protoscoleces per well, the culture medium is FBS-free DMEM medium (containing 1% penicillin-streptomycin), and 200 μL per well.

[0041] The amlodipine in the present application is prepared into a DMSO solution with a concentration of 10 mM for standby. Then the culture well containing the protoscolex is added, so that the final volume of the culture well is 200 μL, and the final concentration of the drug is 60 μM, and a DMSO control group without the drug is set, and the culture plate is cultured in a 37°C, 5% CO2 incubator, and observed and photographed under microscope every 30 minutes.

[0042] 2. Results

[0043] The experimental results are shown in Figure 3 .

[0044] From Figure 3The results show that when amlodipine acts on the protoscolex at a concentration of 60 μM, it can completely kill the protoscolex in only 8 hours. Under a microscope, no signs of activity of the protoscolex can be observed, and compared with the DMSO control group, obvious differences in the structure of the worm body can be seen in the field of view at different magnifications. After amlodipine acts, the refractive property of the protoscolex worm body becomes poor, the worm body shrinks, the edges are not neat, there are signs of shedding of the hook on the protoscolex, the worm body has a tendency to disintegrate, and loses its normal shape. However, after DMSO acts, the protoscolex has obvious signs of activity under a microscope, the edges of the worm body are neat, the surface is smooth, the structure is clear, and the vitality of the worm body is good.

[0045] Example 3

[0046] This example provides the EC of amlodipine for killing Echinococcus multilocularis 50 The concentration (half maximal effective concentration) is calculated by using non-linear regression.

[0047] 1. Materials and methods

[0048] The protoscoleces were obtained by the method mentioned above and cultured in a 96-well cell culture plate. The final concentration of amlodipine was set to be in the range of 10-80 μM (the final concentrations were 10 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 60 μM and 80 μM, respectively). The 10 mM drug stock solution was diluted according to the above concentrations, and then added to the corresponding protoscolex culture wells. The final concentration was the above series of concentrations, and the final volume of the culture well was 200 μL. Each drug concentration for protoscolex was set to be biologically triplicated, and the corresponding volume of DMSO control group was also set. The culture plate was incubated in a 37°C, 5% CO2 incubator for 24 hours, and then the protoscoleces in the culture plate were stained with FDA+PI.

[0049] FDA+PI staining: The protoscoleces in the culture well were washed with PBS for 3 times, the FDA staining solution was diluted with Hanks buffer at a ratio of 1:4000 and added to the culture well, and incubated at 37°C for 10 minutes. After 10 minutes, the FDA staining solution in the culture well was removed, the protoscoleces in the culture well were washed with Hanks buffer for 3 times, and then 2 μL of PI staining solution was added. Incubate at 2-8°C for 5 minutes, remove the PI staining solution in the culture well, resuspend the protoscoleces with Hanks buffer, and take pictures under a fluorescence microscope. If the protoscolex is dead, the protoscolex will be red after PI staining. If the protoscolex is not dead, the protoscolex will be green after FDA staining. Note that the whole process should be operated in the dark.

[0050] EC 50 Concentration statistics: data analysis was performed using GraphPad Prism 9.5, and non-linear regression was used to calculate the EC of amlodipine acting on Echinococcus multilocularis 50 Concentration.

[0051] 2. Results

[0052] The experimental results are shown in Figure 4 and Figure 5 shown.

[0053] Depend on Figure 4 The results showed that after the protoscolex was treated with different concentrations of amlodipine, the images taken using the natural light, red fluorescence and green fluorescence channels of the microscope could clearly distinguish between live and dead protoscolex. When the drug concentration was ≥40μM, most protoscolex emitted red fluorescence, and the protoscolex structure was destroyed under natural light, indicating that the effective concentration of the drug to kill protoscolex was low, and the drug had a clear dose-dependence on the killing effect on protoscolex. Figure 5 The results showed that the EC of amlodipine 50 The concentration was 33.77%±1.529%, which once again showed that low concentrations of amlodipine could effectively kill protoscolex. The death number of protoscolex in the DMSO control group was low, indicating that DMSO had little effect on protoscolex.

Claims

1. Use of amlodipine for the preparation of a medicament for the treatment of echinococcosis, characterized in that, The amlodipine also includes pharmaceutically acceptable salts thereof.

2. Use of amlodipine for the preparation of a medicament for the treatment of echinococcosis according to claim 1, characterized in that, The echinococcosis is alveolar echinococcosis.

3. Use of amlodipine for the preparation of a medicament for the treatment of echinococcosis according to claim 2, characterized in that, The echinococcus includes at least one of its cyst, germinal layer, ascus, vesicle, hydatid fluid or protoscolex.

4. A pharmaceutical composition for treating echinococcosis, characterized by, The pharmaceutical composition includes amlodipine or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable adjuvants.

5. A pharmaceutical composition for the treatment of echinococcosis according to claim 4, characterized in that, The dosage form of the pharmaceutical composition is tablet, granule, powder, capsule, oral liquid, oral, injection, implant or patch.

6. A method for killing Echinococcus in vitro, characterized in that, Amlodipine or pharmaceutically acceptable salts thereof is dissolved in a solvent to prepare a mother liquor, and diluted with the same solvent to obtain a dilute liquor, and then added to the culture system of echinococcus.

7. A method of killing Echinococcus in vitro according to claim 6, wherein, The pharmaceutically acceptable salt of amlodipine is maleate.

8. A method of killing Echinococcus in vitro according to claim 7, wherein, The solvent is DMSO, and the concentration of the dilute liquor is 20-80 μM.

Images