Application of benzyprodil in preparation of medicine for treating echinococcosis

Abstract

The invention discloses an application of benzyprodil in preparation of a medicine for treating echinococcosis. The invention also provides the application of the benprodil or the pharmaceutically acceptable salt thereof or the hydrate of the pharmaceutically acceptable salt thereof in preparation of the medicine for treating the echinococcosis. The invention further provides a pharmaceutical composition for treating the echinococcosis, and the pharmaceutical composition comprises the benprodil or the pharmaceutically acceptable salt of the benprodil or the hydrate of the pharmaceutically acceptable salt of the benprodil, and pharmaceutically acceptable auxiliary materials. Compared with albendazole used for clinically treating the echinococcosis in the field, the benprodil has the advantages that the effect of killing protoscolex in vitro is more remarkable, the dosage is small, the safety is high, the echinococcosis killing effect is faster, and the benprodil has important reference value for developing new echinococcosis treatment drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of the development and application of anti - parasitic drugs, relates to the application of bepridil, and specifically relates to the application of bepridil in the preparation of drugs for treating echinococcosis. Background Art

[0002] Echinococcosis is an important zoonotic parasitic disease caused by the larvae of Echinococcus tapeworms parasitizing in important organs such as the liver and lungs of intermediate hosts. There are 9 recognized species of Echinococcus tapeworms. Among them, the most common, widely distributed, and harmful ones are Echinococcus granulosus and Echinococcus multilocularis. Their larvae cause cystic echinococcosis and alveolar echinococcosis (also known as "worm cancer") in intermediate hosts respectively. The intermediate hosts of cystic echinococcosis are mainly humans and herbivores. The cystic tissue formed by echinococci will cause space - occupying lesions at the parasitic site. The cyst is in the form of a single cyst. As the parasitic time prolongs, the cyst continuously enlarges, compressing the organ and causing organ dysfunction at the same time. If the cyst ruptures under external force, it will trigger an acute allergic reaction or even death in the host, and the spilled protoscoleces will colonize other organs and cause secondary infections. The intermediate hosts of alveolar echinococcosis are mainly humans and rodents. The cyst morphology of alveolar echinococcosis is different from that of cystic echinococcosis. Its cysts grow infiltratively in the parasitic organs and are in the shape of a bunch of grapes. This cyst proliferation mode similar to that of tumors seriously damages the function of the organs. The 10 - year fatality rate of untreated patients after infection can be as high as 94%. At present, the treatment method of echinococcosis is surgical removal of the cyst, and benzimidazole drugs (albendazole and mebendazole) are used as adjuvant treatment. However, these drugs have poor effects in killing the scolices of echinococci, and long - term use will cause adverse liver reactions and other adverse side effects in patients. If the cyst cannot be completely removed by surgery, there is a possibility of recurrence; in addition, benzimidazole drugs are a kind of broad - spectrum anti - parasitic drugs, which mainly achieve the purpose of anti - parasitism by binding to the tubulin of parasites and inhibiting the polymerization and formation of microtubules. At the same time, the function of tubulin in the host will also be inhibited, and long - term use may cause irreversible effects on the host. In short, the current treatment means of echinococcosis mainly rely on surgery, and the role of drugs is very limited. Therefore, there is an urgent need in clinical practice to screen small - molecule compounds for the treatment of echinococcosis that are safe, effective, and low - toxic, and it is also necessary to explore the value of drugs that have been approved by the PDA for the treatment of other diseases in the treatment of echinococcosis to replace benzimidazole drugs. Summary of the Invention

[0003] The object of the present invention is to provide the application of bepridil in the preparation of drugs for treating echinococcosis.

[0004] In the present invention, bepridil is a calcium channel blocker used for antiarrhythmia. This drug inhibits both calcium and sodium currents and has application potential in certain ischemic ventricular arrhythmias. During the screening of drugs against echinococcosis, it was found that bepridil has an effective killing effect on echinococcus.

[0005] The present invention provides the use of bepridil or a pharmaceutically acceptable salt thereof or a hydrate of a pharmaceutically acceptable salt thereof in the preparation of a drug for treating echinococcosis.

[0006] In the present invention, the structural formula of bepridil is as follows:

[0007]

[0008] In the above application, the echinococcosis includes cystic echinococcosis and alveolar echinococcosis.

[0009] In the present invention, the echinococcus is the larva of echinococcus tapeworm, including echinococcus granulosus and / or echinococcus multilocularis;

[0010] The echinococcus tapeworm is one or more of the adults or larvae (echinococcus) of echinococcus granulosus sensu stricto (G1 - G3), echinococcus multilocularis, echinococcus vogeli, echinococcus oligarthrus, echinococcus canadensis (G6 - G10), echinococcus ortleppi (G5), echinococcus equinus (G4), echinococcus felidis, and echinococcus shiquicus.

[0011] In the above application, the pharmaceutically acceptable salts of bepridil are selected from at least one of hydrochloride, nitrate, mesylate, phosphate, citrate, fumarate, sulfate, succinate, tartrate, citrate, hydrobromide, hydroiodide, acetate, lactate, benzenesulfonate, cinnamate, salicylate, malonate, glutarate, and malate.

[0012] In the above application, the echinococcus includes at least one of its cyst, germinal layer, daughter cyst, grand - daughter cyst, cyst fluid, and protoscolex.

[0013] In the above application, the dosage form of the drug is selected from at least one of tablets, granules, powders, capsules, oral liquids, buccal tablets, injections, implants, and patches.

[0014] The present invention also provides a pharmaceutical composition for treating echinococcosis, which comprises bepridil or a pharmaceutically acceptable salt thereof or a hydrate of a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0015] In the above pharmaceutical composition, the dosage form of the pharmaceutical composition is selected from at least one of tablets, granules, powders, capsules, oral liquids, buccal tablets, injections, implants, and patches.

[0016] The present invention further provides a method for killing Echinococcus in vitro, which includes dissolving bepridil or its pharmaceutically acceptable salt or hydrate of its pharmaceutically acceptable salt in a solvent to prepare a mother liquor containing bepridil or its pharmaceutically acceptable salt, and adding the diluted mother liquor of bepridil or its pharmaceutically acceptable salt to the culture system of Echinococcus for culturing to kill Echinococcus.

[0017] In the above method, the concentration of the diluted mother liquor of bepridil or its pharmaceutically acceptable salt is 30 - 250 μM, preferably 30 - 100 μM;

[0018] The pharmaceutically acceptable salt of bepridil is the hydrochloride salt of bepridil, and the corresponding solvent is DMSO.

[0019] In the above method, the culture conditions are culturing in an incubator at 37°C and 5% CO₂.

[0020] The present invention has the following beneficial effects:

[0021] Compared with albendazole, which is used in the field for clinical treatment of echinococcosis, bepridil of the present invention has a more significant effect on killing protoscoleces in vitro, with less dosage, high safety, a more rapid effect on killing Echinococcus, and has important reference value for the development of new drugs for the treatment of echinococcosis. Description of the Drawings

[0022] Figure 1 It is an optical microscope (40×) image of protoscoleces of Echinococcus multilocularis after 24 - hour in vitro treatment with 125 μM bepridil, albendazole and an equal volume of DMSO in Example 1. The first - row pictures are images of non - stained protoscoleces, and the second - row pictures are trypan blue - stained pictures.

[0023] Figure 2 It is a statistical chart of the significance of the insecticidal effect of protoscoleces of Echinococcus multilocularis after 24 - hour in vitro treatment with 125 μM bepridil, albendazole and an equal volume of DMSO in Example 1.

[0024] Figure 3 It is a diagram of the morphological changes of protoscoleces of Echinococcus multilocularis after 6 - hour in vitro treatment with 40 μM bepridil and an equal volume of DMSO in Example 2 (magnification multiples are 50×, 100×, 200× and 400× respectively).

[0025] Figure 4 It is a natural light and red fluorescence image of protoscoleces of Echinococcus multilocularis after 24 - hour in vitro treatment with bepridil at a concentration of 1 - 80 μM and an equal volume of DMSO in Example 3, Figure 4In the text, BPD is bepridil; BPD PI is PI staining after the protoscoleces are treated with bepridil; DMSO is dimethyl sulfoxide; DMSO PI is PI staining after the protoscoleces are treated with dimethyl sulfoxide.

[0026] Figure 5 EC of bepridil against Echinococcus multilocularis in Example 3 50 Concentration statistical results. Specific implementation manners

[0027] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0028] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0029] Example 1:

[0030] This example provides a method for killing Echinococcus multilocularis with bepridil. The specific experimental operation steps are as follows:

[0031] 1 Materials and methods

[0032] 1.1 Construction of Echinococcus multilocularis mouse infection model: Echinococcus multilocularis is obtained from the abdominal cavity of BALB / c mice preserved in the laboratory. The protoscoleces are washed in PBS buffer containing 1% penicillin-streptomycin until the solution where the protoscoleces are located is clear. Then the protoscoleces solution is thoroughly resuspended with a Pasteur pipette. 10 μL of the protoscoleces resuspension is pipetted onto a glass slide (3 drops are taken as biological replicates). The number of protoscoleces in 3 drops of 10 μL resuspension is counted under an optical microscope and the average value is taken. According to the counting result, the number of protoscoleces in the resuspension is adjusted to 10 protoscoleces per μL. 200 μL of the protoscoleces resuspension (about 2000 protoscoleces) is taken and intraperitoneally injected into BALB / c mice (specifically, 6-8-week-old BALB / c mice, purchased from the Experimental Animal Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, with a body weight of about 20 g), thus completing the construction of the Echinococcus multilocularis mouse infection model. After 2 months of infection, the hydatid cyst tissue and protoscoleces are obtained from the abdominal cavity for efficacy evaluation.

[0033] 1.2 Acquisition of protoscoleces and drug action in vitro: Prepare DMEM medium, Pasteur pipettes, culture dishes, 50-ml centrifuge tubes, glass slides, alcohol cotton, 75% alcohol, surgically sterile instruments, PBS buffer, and beakers with sieves; after decapitating BALB / c mice, soak them in disinfectant alcohol for surface disinfection, then open the abdominal cavity to remove the cysticercus tissue of Echinococcus multilocularis onto the sieve beaker, cut the cysticercus tissue into pieces with scissors, and wash the protoscoleces in the broken cyst into the beaker with PBS buffer. Repeat this several times to allow the protoscoleces to fully drain. Remove the sieve from the beaker, let it stand for 5 minutes, then aspirate and discard the upper waste liquid in the beaker. Then aspirate the sedimented protoscoleces into a 6-cm culture dish, add PBS buffer, evenly disperse the protoscoleces, and let it stand for 5 minutes. Rotate the culture dish clockwise to quickly gather the protoscoleces to the center of the dish. Aspirate and discard the surrounding liquid, then add PBS buffer again and disperse the protoscoleces. Repeat the above operation 4-5 times until the liquid is clear after standing for several minutes. Aspirate the protoscoleces in the center into a 50-ml centrifuge tube, let it stand, and only retain 10 ml of liquid. After pipetting and mixing the protoscoleces in the centrifuge tube, aspirate 3 drops of 10-μl liquid onto a glass slide, count under a microscope, and take the average value. Finally, adjust the liquid volume to ensure that there are 100 protoscoleces in every 10 μl of liquid. Add 190 μL of DMEM culture medium to each well of a 96-well cell culture plate, then mix the protoscoleces and add 10 μl of the protoscolex mixture to each well, and culture overnight. Set 6 concentrations of bepridil (dissolved in dimethyl sulfoxide): 250 μM, 125 μM, 75 μM, 50 μM, 30 μM, 10 μM, with 3 biological replicates for each concentration. At the same time, set the corresponding volume of DMSO as a control and a blank control (without drug treatment), and set albendazole as a positive control.

[0034] 1.3 Observation and staining of the killing effect of protoscoleces: Wash the protoscoleces in the culture plate 3 times with PBS buffer, observe the morphology and viability of the protoscoleces under a microscope, and observe each well for 1 minute to determine whether the protoscoleces are active and whether they have disintegrated, initially judging the killing effect of the drug on the protoscoleces. Then add 4 μL of 1% trypan blue staining solution to each well and stain for 3 minutes. Wash 5 times with PBS buffer until the staining solution is completely washed away and there is no obvious background color. Observe under a microscope whether the protoscoleces are stained. If they are stained blue and the protoscoleces are immobile, it can be determined that the protoscoleces have been killed.

[0035] 1.4 Statistics of the killing effect of drugs on protoscoleces: Take pictures of the microscopic examination results and count the mortality rate of the protoscoleces. Mortality rate (%) = (number of dead protoscoleces in the drug-treated group - number of dead protoscoleces in the control group) / (total number of protoscoleces in the culture well - number of dead protoscoleces in the control group) × 100%.

[0036] 1.5 Differential statistical analysis: GraphPad Prism 8 was used for data analysis. The lethality of the drug against protoscoleces was expressed as the mean ± standard deviation (SD), and then one-way ANOVA (analysis of variance) was selected for significant difference analysis. Note: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0037] 1.6 Reagents: The compounds bepridil, albendazole, and DMSO used in this experiment were all purchased from MCE Company.

[0038] 2 Results

[0039] The lethal effects of bepridil and albendazole on the protoscoleces of Echinococcus multilocularis are shown in Figure 1 、 Figure 2 and Table 1.

[0040] Table 1 Lethality of drugs at different concentrations on Echinococcus multilocularis in vitro

[0041]

[0042] From Figure 1 and Figure 2 The results showed that after treating Echinococcus multilocularis with 125 μM bepridil for 48 hours and observing the protoscoleces under a microscope, it was found that the structure of the worms was damaged and there was no sign of activity, indicating that the drug exerted a lethal effect, and all protoscoleces were stained with trypan blue; while the protoscoleces in the albendazole and DMSO control groups had intact structures, and it was observed under a microscope that most of the protoscoleces had signs of activity, their viability was not significantly affected and almost none of them were stained with trypan blue, indicating that the lethal effect of albendazole on protoscoleces was not obvious. The results in Table 1 showed that when the concentration of bepridil was 30 - 250 μM, the lethality rate against protoscoleces reached 100%, and when the drug concentration was as low as 10 μM, the lethality rate of protoscoleces was 32.5%, showing an obvious killing effect on protoscoleces, and the effect was significantly better than that of albendazole; for albendazole, the overall effect of killing protoscoleces was poor, and the change in drug concentration did not significantly change its killing effect on protoscoleces.

[0043] Example 2:

[0044] This example provides the microscopic structural changes of bepridil in killing Echinococcus multilocularis. The specific experimental operation steps are as follows:

[0045] 1 Materials and Methods

[0046] After decapitating the BALB / c mice infected with Echinococcus multilocularis modeled in Example 1 of the present invention, disinfect the body surface, cut open the abdominal cavity, take out the echinococcal cyst tissue and place it on a beaker tied with a sieve. After cutting the cyst tissue into pieces, rinse it with PBS buffer containing 1% penicillin-streptomycin until the protoscoleces leak into the beaker. Then, rinse the protoscoleces in the beaker repeatedly according to the above method. Transfer the rinsed protoscoleces to a 50 ml centrifuge tube for counting, and then culture the protoscoleces in a 96-well cell culture plate, with 100 protoscoleces in each well. The culture medium used is DMEM medium without FBS (containing 1% penicillin-streptomycin), 200 μL per well.

[0047] Prepare a DMSO solution of bepridil in the present invention at a concentration of 10 mM for standby. Then add it to the culture wells containing protoscoleces to make the final volume of the culture wells 200 μL and the final concentration of the drug 40 μM. At the same time, set up an equal-volume DMSO control group without the drug. The culture plate is cultured in an incubator at 37°C and 5% CO2, and observed under a microscope and photographed every 30 minutes.

[0048] 2 Results

[0049] The experimental results are shown in Figure 3 as follows.

[0050] As Figure 3 the results show that when bepridil acts on protoscoleces at a concentration of 40 μM, it only takes 6 hours to completely kill the protoscoleces. Under the microscope, there is no sign of activity of the protoscoleces. Compared with the DMSO control group, obvious differences in the worm body structure can be seen in the fields of view at different magnifications. After the action of bepridil, the refractive index of the protoscoleces becomes worse, the worm body shrinks, the edge is uneven, there is a sign of shedding of the barbs on the protoscoleces, and the worm body has a tendency to disintegrate and loses its normal shape; while the protoscoleces after the action of DMSO show obvious signs of activity under the microscope, the edge of the worm body is neat, the surface is smooth, the structure is clear, and the vitality of the worm body is good.

[0051] Example 3:

[0052] This example provides the EC 50 concentration (maximum half-effective concentration) of bepridil in killing Echinococcus multilocularis. The specific experimental operation steps are as follows:

[0053] 1 Materials and Methods

[0054] The protoscolices were obtained by the method mentioned above and cultured in a 96-well cell culture plate. The final concentration range of bepridil was set to 1-80 μM (the final concentrations were 1 μM, 5 μM, 10 μM, 12 μM, 15 μM, 18 μM, 20 μM, 25 μM, 40 μM, and 80 μM). The 10 mM drug stock solution was diluted according to the above concentrations and then added to the corresponding protoscolices culture wells. The final concentration was the above series of concentrations, and the final volume of the culture well was 200 μL. Three biological replicates were set for each drug concentration of the protoscolices, and a DMSO control group with the corresponding volume was also set. The culture plate was cultured in an incubator at 37 °C and 5% CO2 for 24 hours, and then the protoscolices in the culture plate were stained with PI.

[0055] PI staining: Wash the protoscolices in the culture wells 3 times with PBS, add 100 μL of HBSS buffer to the culture wells, then add 2 μL of PI staining solution, incubate at 2-8 °C for 5 minutes, aspirate the PI staining solution in the culture wells, and resuspend the protoscolices with HBSS buffer. Take pictures and record under a fluorescence microscope. If the protoscolices are dead, they will emit red fluorescence after PI staining. Note that the whole process should be carried out in the dark.

[0056] EC 50 Concentration statistics: Use GraphPad Prism 8 for data analysis and calculate the EC 50 concentration of bepridil acting on Echinococcus multilocularis using non-linear regression.

[0057] 2 Results

[0058] The experimental results are shown in Figure 4 and Figure 5 .

[0059] From Figure 4 the results, it can be seen that after the protoscolices were treated with different concentrations of bepridil, the live and dead protoscolices could be clearly distinguished by the pictures taken under the natural light and red fluorescence channels of the microscope. When the drug concentration ≥ 18 μM, most of the protoscolices emitted red fluorescence, and the worm body structure of the protoscolices was damaged under natural light, indicating that the effective concentration of this drug to kill the protoscolices was low, and at the same time, the killing of the protoscolices by the drug had an obvious dose-dependence; from Figure 5 the results, it can be seen that the EC 50 concentration of bepridil was 17.21 ± 0.37, which once again indicated that a low concentration of bepridil could effectively kill the protoscolices. The number of dead protoscolices in the DMSO control group was low, indicating that DMSO had little effect on the protoscolices.

Claims

1. Use of bepridil or a pharmaceutically acceptable salt thereof or a hydrate of a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating echinococcosis.

2. The use according to claim 1, characterized in that: The echinococcosis includes cystic echinococcosis and alveolar echinococcosis.

3. The use according to claim 1 or 2, characterized in that: The pharmaceutically acceptable salt of bepridil is selected from at least one of hydrochloride, nitrate, methanesulfonate, phosphate, citrate, fumarate, sulfate, succinate, tartrate, citrate, hydrobromide, hydroiodide, acetate, lactate, tosylate, cinnamate, salicylate, malonate, glutarate and malate.

4. The use according to any one of claims 1 to 3, characterized in that: The echinococcosis includes at least one of its cyst, germinal layer, daughter cyst, granddaughter cyst, cyst fluid and protoscolex.

5. The use according to any one of claims 1 to 4, characterized in that: The dosage form of the drug is selected from at least one of tablets, granules, powders, capsules, oral liquids, lozenges, injections, implants, and patches.

6. A pharmaceutical composition for treating echinococcosis, characterized in that: The pharmaceutical composition comprises bepridil or a pharmaceutically acceptable salt thereof or a hydrate of a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

7. The pharmaceutical composition according to claim 6, characterized in that The dosage form of the pharmaceutical composition is selected from at least one of tablets, granules, powders, capsules, oral liquids, lozenges, injections, implants, and patches.

8. A method for killing echinococcosis in vitro, characterized in that: The method comprises dissolving bepridil or a pharmaceutically acceptable salt thereof or a hydrate of a pharmaceutically acceptable salt thereof in a solvent to prepare a mother solution containing bepridil or a pharmaceutically acceptable salt thereof, and adding the diluted mother solution of bepridil or a pharmaceutically acceptable salt thereof to a culture system of echinococcosis for culture to kill the echinococcosis.

9. The method according to claim 8, characterized in that The concentration of the mother solution of bepridil or its pharmaceutically acceptable salt after dilution is 30 to 250 μM; The pharmaceutically acceptable salt of bepridil is bepridil hydrochloride, and the corresponding solvent is DMSO.

10. The method according to claim 8 or 9, characterized in that: The culture conditions are culturing in an incubator at 37° C. and 5% CO 2 .

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