Application of ceritinib in preparation of medicine for treating echinococcosis
By combining with ALK fusion protein, ceritinib is prepared into various dosage forms for the treatment of echinococcosis, solving the side effects and recurrence risks of existing treatments and achieving efficient and safe treatment of echinococcosis.
Patent Information
- Application Number
- CN202510858668.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-25
- Publication Date
- 2025-10-10
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of development and application of antiparasitic drugs, and relates to the application of ceritinib, and in particular to the application of ceritinib in the preparation of a drug for treating echinococcosis. Background Art
[0002] Currently, the treatment for echinococcosis is surgical removal of the cysts, combined with adjuvant therapy using benzimidazoles such as albendazole or mebendazole. However, these drugs are ineffective in killing echinococcosis, and long-term use can cause adverse liver reactions and other adverse side effects in patients. Furthermore, if the cysts cannot be completely removed surgically, there is a possibility of recurrence. Furthermore, benzimidazoles are broad-spectrum antiparasitic drugs that primarily inhibit the polymerization and formation of microtubules by binding to the parasite's microtubules. However, the function of microtubules in the host is also inhibited, and long-term use may cause irreversible effects on the host. Therefore, there is an urgent need to screen for safe, effective, and low-toxic small molecule compounds for the treatment of echinococcosis. Summary of the Invention
[0003] The present invention aims to provide the use of ceritinib in preparing a medicament for treating echinococcosis.
[0004] Ceritinib, described in the present invention, is a highly selective anaplastic lymphoma kinase (ALK) inhibitor that precisely binds to the ALK fusion protein and, by occupying its ATP binding site, inhibits ALK kinase activity, showing potential for application in cancer treatment. During the screening of anti-echinococcosis drugs, the applicants made the groundbreaking discovery that ceritinib effectively kills echinococcosis larvae and can be used as a core pharmacological agent in the preparation of various pharmaceutical dosage forms for the treatment of echinococcosis, such as tablets, capsules, and injections. This provides new drug options and treatment strategies for echinococcosis.
[0005] The structural formula of Ceritinib is as follows:
[0006]
[0007] In the application of the present invention, the ceritinib also includes a pharmaceutically acceptable salt thereof or a hydrate of a pharmaceutically acceptable salt thereof.
[0008] Furthermore, the echinococcosis is cystic echinococcosis or alveolar echinococcosis.
[0009] The echinococcosis is the larvae of Echinococcus, including Echinococcus granulosus and Echinococcus multilocularis; the Echinococcus is one or more of the adults or larvae (echinococcosis) of Echinococcus granulosus sensu stricto (G1-G3), Echinococcus multilocularis, Echinococcus vorkinensis, Echinococcus oligosegmentosus, Echinococcus canadensis (G6-G10), Echinococcus ostreatus (G5), Echinococcus equi (G4), Echinococcus leonina and Echinococcus spp.
[0010] The echinococcosis includes at least one of its cyst, germinal layer, daughter cyst, grand cyst, cyst fluid or protoscolex.
[0011] Furthermore, the dosage form of the drug is selected from at least one of tablets, granules, powders, capsules, oral liquids, buccal preparations, injections, implants or patches.
[0012] The present invention also provides a pharmaceutical composition for treating echinococcosis, which comprises ceritinib or a pharmaceutically acceptable salt thereof or a hydrate of a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
[0013] The present invention further provides a method for killing echinococcosis in vitro, which comprises dissolving the ceritinib or a pharmaceutically acceptable salt thereof or a hydrate of a pharmaceutically acceptable salt thereof in a solvent to prepare a mother liquor, diluting the mother liquor with the same solvent to obtain a dilution, and then adding the dilution to a culture system of echinococcosis to kill the echinococcosis.
[0014] Furthermore, the pharmaceutically acceptable salt of ceritinib is hydrochloride. The solvent is DMSO, and the concentration of the diluent is 10 to 80 μM;
[0015] Furthermore, the culture conditions are 37° C. and 5% CO 2 .
[0016] The present invention has the following beneficial effects:
[0017] The present invention unexpectedly discovered that the compound ceritinib is significantly effective in killing protoscolex in vitro. Results from a method for killing Echinococcus multilocularis showed that at the highest experimental concentration of 80 μM, albendazole had a lethality rate of only 2.5% ± 0.5% against protoscolex. However, at the lowest experimental concentration of 10 μM, the ceritinib of the present invention achieved a lethality rate of 16.00% ± 2.75% against protoscolex, far exceeding the lethality rate of albendazole at the highest experimental concentration. Furthermore, at concentrations of 25 to 80 μM, the lethality rate against protoscolex can reach as high as 100%. Experiments on microstructural changes in the killing of Echinococcus multilocularis by ceritinib showed that at a concentration of 35 μM, ceritinib completely killed protoscolex in just 8 hours. Therefore, compared with albendazole, which is used in the field for clinical adjuvant treatment of echinococcosis, the ceritinib of the present invention has a more significant effect in killing protoscolex in vitro, requires less dosage, is safer, and has a faster echinococcosis killing effect, which has important reference value for the development of new drugs for the treatment of echinococcosis. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 These are optical microscopic images (40×) of the protoscolex of multilocularis treated with 60 μM ceritinib, albendazole, and an equal volume of DMSO for 24 hours in vitro, as described in Example 1, along with untreated blank controls. The first row of images shows unstained protoscolex, while the second row shows images stained with FDA (fluorescein diacetate) and propidium iodide (PI). In the following figures, D28 refers to ceritinib, ABZ to albendazole, and DMSO to dimethyl sulfoxide.
[0019] Figure 2 This is a statistical graph showing the insecticidal effect of 60 μM ceritinib, albendazole and an equal volume of DMSO on protoscolex multilocularis in vitro for 24 hours in Example 1.
[0020] Figure 3 These are pictures showing the morphological changes of Echinococcus multilocularis after 8 hours of in vitro treatment with 35 μM ceritinib and an equal volume of DMSO in Example 2 (magnifications are 50×, 100×, 200×, and 400×, respectively).
[0021] Figure 4 The following are the microscopic bright field images and red-green combined fluorescence images of the worm bodies after 24 hours of in vitro treatment of Echinococcus multilocularis with 10-80 μM concentration of ceritinib and an equal volume of DMSO in Example 3; D28 FDA+PI is a combined fluorescence image of FDA and PI staining after the protoscolex is treated with ceritinib; DMSO FDA+PI is a combined fluorescence image of FDA and PI staining after the protoscolex is treated with dimethyl sulfoxide.
[0022] Figure 5 Example 3: Effect of Ceritinib on EC of Echinococcus multilocularis 50Concentration statistics results. DETAILED DESCRIPTION
[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0024] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0025] Example 1
[0026] This example provides a method for killing Echinococcus multilocularis with Ceritinib. The specific experimental steps are as follows:
[0027] 1 Materials and Methods
[0028] 1.1 Construction of multilocular Echinococcus infection model in mice: Multilocular Echinococcus cysts were obtained from the peritoneal cavity of BALB / c mice maintained in the laboratory. The protoscoleci were washed in PBS buffer with 1% penicillin-streptomycin until the solution containing the protoscoleci was clear. The protoscoleci solution was then thoroughly resuspended with a Pasteur pipette. 10 μL of the resuspended protoscoleci solution was aspirated and dropped onto a glass slide (3 drops were aspirated as biological replicates). The number of protoscoleci in 3 drops of 10 μL of the resuspended solution was counted under an optical microscope and the average value was calculated. The number of protoscolex in the resuspension was adjusted to 10 protoscolex per microliter based on the counting results. After aspirating 200 microliters of the protoscolex resuspension (approximately 2000 protoscolex), it was 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, weighing approximately 20 g), to complete the construction of the multilocular Echinococcus mouse infection model. Two months after infection, the Echinococcus cyst tissue and protoscolex were obtained from the abdominal cavity for drug efficacy evaluation.
[0029] 1.2 Obtaining protoscolex and in vitro drug effects: Prepare DMEM culture medium, Pasteur pipette, culture dish, 50ml centrifuge tube, slide, alcohol cotton, 75% alcohol, autoclaved surgical instruments, PBS buffer and beaker with mesh; BALB / c mice were dislocated in the neck and immersed in disinfectant alcohol for surface disinfection, then the abdominal cavity was opened to remove the multilocular echinococcosis cyst tissue and put it on the mesh beaker, then the cyst tissue was cut into pieces with scissors and the protoscolex in the broken cyst was flushed into the beaker with PBS buffer, and the flushing was repeated several times to allow the protoscolex to drain fully, the mesh on the beaker was removed and the upper layer of waste liquid in the beaker was discarded after standing for 5 minutes, and the settled protoscolex was sucked into a 6cm culture dish, PBS buffer was added and the protoscolex was evenly blown away and the dish was left to stand for 5 minutes, the dish was rotated clockwise to make the protoscolex quickly gather in the center of the dish, the surrounding liquid was discarded and PBS buffer was added to disperse the protoscolex, and the dish was rested for several minutes. Repeat the above operation 4-5 times until the liquid is clear, and draw the protoscolex in the center into a 50ml centrifuge tube. After standing, only 10ml of liquid is retained. After blowing and mixing the protoscolex in the centrifuge tube, draw 3 drops of 10 microliters of liquid onto a slide, and then count under a microscope and take the average value. Finally, increase or decrease the amount of liquid so that each 10 microliters of liquid contains 100 protoscolex. Add 190μL of DMEM culture medium to each well of the 96-well cell culture plate, and then After mixing the protoscolex, 10 μL of the protoscolex mixture was added to each well and cultured overnight. Eight concentrations of ceritinib (ceritinib dissolved in dimethyl sulfoxide) were set: 80 μM, 60 μM, 40 μM, 35 μM, 30 μM, 25 μM, 20 μM, and 10 μM, with three biological replicates for each concentration. At the same time, the corresponding volume of DMSO was set as a control and a blank control (no drug treatment) was set. In addition, albendazole was set as a positive control.
[0030] 1.3 Observation and staining of the killing effect of protoscolex: The protoscolex in the culture plate was washed three times with PBS buffer, and the morphology and activity of the protoscolex were observed under a microscope. Each well was observed for 1 minute to determine whether the protoscolex was active and whether it was disintegrated, so as to preliminarily judge the killing effect of the drug on the protoscolex. Then, the FDA dye solution was diluted with Hanks buffer at a ratio of 1:4000 and added to the culture wells, and incubated at 37°C for 10 minutes. After 10 minutes, aspirate the FDA stain in the culture well, wash the protoscolex in the culture wells 3 times with Hanks buffer, then add 2 μL of PI stain, incubate at 2-8°C for 5 minutes, aspirate the PI stain in the culture wells and resuspend the protoscolex with Hanks buffer, take pictures and record under a fluorescence microscope. If the protoscolex is dead, it will emit red fluorescence after PI staining. If it is not dead, it will emit green fluorescence after FDA staining. Note that the whole process should be kept away from light.
[0031] 1.4 Statistics of the killing effect of drugs on protoscolex: The microscopic examination results were photographed and the mortality rate of protoscolex was calculated. Mortality rate (%) = (number of dead protoscolex in the drug group - number of dead protoscolex in the control group) / (total number of protoscolex in the culture well - number of dead protoscolex in the control group) × 100%.
[0032] 1.5 Statistical Analysis: Data were analyzed using GraphPad Prism 9.5. The lethality of the drugs against protoscolex is expressed as mean ± standard deviation (SD). Significant differences were analyzed using one-way ANOVA. Notes: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
[0033] 1.6 Reagents: The compounds used in this experiment, ceritinib, albendazole, and DMSO, were purchased from MCE.
[0034] 2. Results
[0035] The lethality of ceritinib and albendazole against multilocular protoscolex is shown in Figure 1 、 Figure 2 and as shown in Table 1.
[0036] Table 1 The lethality of different concentrations of drugs against Echinococcus multilocularis in vitro
[0037]
[0038] Depend on Figure 1 and Figure 2 The results showed that after 24 hours of treatment with 60μM ceritinib on multilocular Echinococcus, the protoscolex was observed under a microscope and found that the worm structure was destroyed and there was no sign of activity, indicating that the drug had exerted a lethal effect. All protoscolex were stained by PI and none were stained by FDA. In contrast, the protoscolex of the albendazole and DMSO control groups had intact worm structures. Under the microscope, most protoscolex showed signs of activity, their vitality was not significantly affected, and they were almost not stained by PI, indicating that albendazole had no obvious lethal effect on protoscolex. The results in Table 1 show that when the effective concentration of ceritinib is 25-80 μM, the lethality of ceritinib against protoscolex reaches 100%. When the drug concentration is as low as 20 μM, the lethality of ceritinib against protoscolex is 38.48%±1.64%, showing a significant protoscolex killing effect, which is significantly better than albendazole. For albendazole, the overall effect of killing protoscolex is poor, and the change of drug concentration does not significantly change its protoscolex killing effect.
[0039] Example 2
[0040] This example provides microstructural changes in the killing of Echinococcus multilocularis by Ceritinib. The specific experimental steps are as follows:
[0041] 1. Materials and Methods
[0042] BALB / c mice infected with multilocular Echinococcus modeled in Example 1 of the present invention were dislocated and the body surface was disinfected. The abdominal cavity was cut open and the cyst tissue of the Echinococcus cyst was removed and placed in a beaker with a sieve. The cyst tissue was chopped and rinsed with PBS buffer containing 1% penicillin-streptomycin until the protoscolex leaked into the beaker. The protoscolex in the beaker was then repeatedly rinsed according to the above method. The rinsed protoscolex was transferred to a 50 ml centrifuge tube for counting, and then the protoscolex was cultured in a 96-well cell culture plate, with 100 protoscolex per well. The culture medium used was FBS-free DMEM medium (containing 1% penicillin-streptomycin), 200 μL per well.
[0043] Ceritinib of the present invention was prepared into a 10 mM DMSO solution for later use. The solution was then added to culture wells containing protoscolex to a final volume of 200 μL and a final drug concentration of 60 μM. A control group containing an equal volume of DMSO without drug was also prepared. The culture plates were incubated in a 37°C, 5% CO2 incubator and observed under a microscope every 30 minutes, with photographs taken.
[0044] 2. Results
[0045] The experimental results are shown in Figure 3 As shown. Figure 3 The results showed that when ceritinib acted on protoscolex at a concentration of 60 μM, it only took 8 hours to completely kill the protoscolex. No signs of activity were observed in the protoscolex under a microscope. Compared with the DMSO control group, obvious differences in the worm structure could be seen in the fields of view at different magnifications. After the action of ceritinib, the refractive index of the protoscolex became worse, the worm body shrank, the edges were uneven, the hooks on the protoscolex showed signs of falling off, the worm body had a tendency to disintegrate, and lost its normal morphology; while after the action of DMSO, the protoscolex showed obvious signs of activity under a microscope, the worm body had neat edges, a smooth surface, a clear structure, and good worm body vitality.
[0046] Example 3
[0047] This example provides the EC of Ceritinib killing multilocular Echinococcus 50 Concentration (maximum half effective concentration), the specific experimental steps are as follows:
[0048] 1. Materials and Methods
[0049] Protoscolex were obtained according to the method mentioned above and cultured in 96-well cell culture plates. The final concentration range of ceritinib was set to 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. The final volume of the culture well was 200 μL. Three biological wells were set for each drug action concentration of the protoscolex. At the same time, a DMSO control group of the corresponding volume was set. The culture plate was cultured in an incubator at 37° C. and 5% CO2 for 24 hours, and then the protoscolex in the culture plate was stained with FDA+PI.
[0050] FDA+PI staining: Wash the protoscolex in the culture wells three times with PBS, dilute the FDA stain with Hanks buffer at a ratio of 1:4000 and add it to the culture wells. Incubate at 37°C for 10 minutes. After 10 minutes, aspirate and discard the FDA stain in the culture wells. Wash the protoscolex in the culture wells three times with Hanks buffer, then add 2μL of PI stain. Incubate at 2-8°C for 5 minutes. Aspirate and discard the PI stain in the culture wells and resuspend the protoscolex with Hanks buffer. Take photos under a fluorescence microscope. If the protoscolex is dead, it will emit red fluorescence after PI staining. If the protoscolex is not dead, it will emit green fluorescence after FDA staining. Note that the entire process should be kept away from light.
[0051] EC 50 Concentration statistics: Data analysis was performed using GraphPad Prism 9.5, and nonlinear regression was used to calculate the EC of ceritinib against Echinococcus multilocularis. 50 concentration.
[0052] 2. Results
[0053] The experimental results are shown in Figure 4 and Figure 5 As shown. Figure 4 The results showed that after different concentrations of ceritinib acted on protoscolex, 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 ≥25 μM, 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-dependency in killing protoscolex. Figure 5 The results showed that the EC of ceritinib 50 The concentration was 20.65%±1.315%, which once again showed that low concentrations of ceritinib can 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 ceritinib in the preparation of a medicament for treating echinococcosis, characterized in that: The ceritinib also includes its pharmaceutically acceptable salt or salt hydrate; the structural formula of the compound ceritinib is shown below:
2. The use of ceritinib according to claim 1 in the preparation of a medicament for treating echinococcosis, wherein: The echinococcosis is cystic echinococcosis or alveolar echinococcosis.
3. The use of ceritinib according to claim 2 in the preparation of a medicament for treating echinococcosis, wherein: The echinococcosis is at least one of its cyst, germinal layer, daughter cyst, grand cyst, cyst fluid or protoscolex.
4. A pharmaceutical composition for treating echinococcosis, characterized in that: The invention comprises ceritinib or a pharmaceutically acceptable salt or salt hydrate thereof, and a pharmaceutically acceptable excipient.
5. A pharmaceutical composition for treating echinococcosis according to claim 4, characterized in that: The dosage form of the pharmaceutical composition is tablets, granules, powders, capsules, oral liquids, buccal preparations, injections, implants or patches.
6. A method for killing echinococcosis in vitro, characterized in that: Ceritinib or a pharmaceutically acceptable salt or salt hydrate thereof is dissolved in a solvent to prepare a mother solution, which is diluted with the same solvent to obtain a dilution, and then added to a culture system of echinococcosis to kill the echinococcosis.
7. The method for killing echinococcosis in vitro according to claim 6, characterized in that: The pharmaceutically acceptable salt of ceritinib is hydrochloride.
8. The method for killing echinococcosis in vitro according to claim 7, characterized in that: The solvent is DMSO, and the dilution concentration is 10-80 μM.
9. The method for killing echinococcosis in vitro according to any one of claims 6 to 8, characterized in that: The culture conditions are 37° C. and 5% CO 2 .