Application of Apoptozole in preparation of medicine for treating echinococcosis multilocularis

Abstract

The invention discloses an application of Apoptozole in preparation of a medicine for treating echinococcosis multilocularis, and belongs to the field of biomedicine. According to the application disclosed by the invention, the Apoptozole can be used for treating and / or preventing the echinococcosis multilocularis, has a remarkable killing effect on the protoscolex of the echinococcosis multilocularis and the vesicle stage of the continuous period in vitro, and can be used for destroying tissue structures such as a head hook, a sucking disc and microvillus of the protoscolex, so that the vesicle collapse in the continuous period is caused, and the echinococcosis multilocularis can be prevented. In addition, in-vivo protoscolex of a mouse secondary infection model with the alveolar echinococcosis can be remarkably inhibited from developing towards cysts, and the infection rate and the liver transfer rate are both reduced. Therefore, the Apoptozole can be used as a medicine for treating the echinococcosis multilocularis.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and particularly relates to application of Apoptozole in preparing a medicine for treating multilocular echinococcosis. Background Art

[0002] Alveolar echinococcosis (AE) is a disease caused by Echinococcus multilocularis,Em ) parasitizes in human organs and is a serious parasitic disease, also known as alveolar hydatid disease. The primary lesions of the disease are almost always located in the liver, with malignant invasive growth, and can also spread to other tissues and organs, such as the lungs, brain and spine, which may also be affected. It is commonly known as "worm cancer" and is highly lethal. In recent years, surgical resection (including liver resection, liver transplantation and autologous liver transplantation) is currently the preferred method for clinical treatment of multilocular echinococcosis, but for AE patients in the terminal stage or without surgical indications, drugs are required for adjuvant treatment. At present, the first choice for clinical treatment of multilocular echinococcosis is albendazole tablets or liposomes. However, the drug level of albendazole reaching the lesion is insufficient, the bioavailability is less than 5%, and it is accompanied by a variety of adverse toxic and side effects, resulting in poor efficacy and limited use. Therefore, there is an urgent need to discover or develop new effective drugs for the treatment of multilocular echinococcosis.

[0003] Apoptozole (Apoptosis Activator VII) can directly induce programmed cell death of tumor cells by activating the caspase-dependent apoptosis pathway, and can also promote apoptosis by making tumor cells unable to repair misfolded proteins caused by stress or treatment damage; at the same time, Apoptozole has been shown in a mouse model with cancer cell xenografts to significantly inhibit tumor growth in vivo without affecting the vitality of the mice. Combined treatment with Apoptozole can also sensitize chemotherapy and radiotherapy or photothermal therapy to inhibit tumor growth. However, whether Apoptozole is effective in treating multilocular echinococcosis, also known as "parasitic liver cancer," has not yet been reported. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to treat and / or prevent multilocular echinococcosis.

[0005] In order to solve the above technical problems, the present invention first provides any of the following applications: 1. Use of apoptozole in the preparation of a drug for treating and / or preventing multilocular echinococcosis; 2. The use of apoptozotocin in the treatment and / or prevention of multilocular echinococcosis; 3. Use of Apoptozole in the preparation of drugs for reducing infection or metastasis of Echinococcus multilocularis; 4. The use of apoptozole in reducing infection or metastasis of Echinococcus multilocularis; 5. Application of Apoptozole in the preparation of products for inhibiting the development of Echinococcus multilocularis; 6. Application of Apoptozole in inhibiting the development of Echinococcus multilocularis; 7. Application of Apoptozole in the preparation of products for killing Echinococcus multilocularis; 8. The application of Apoptozole in killing Echinococcus multilocularis; 9. Application of Apoptozole in the preparation of products for reducing multilocular echinococcosis lesions; 10. The use of apoptozotocin in reducing the lesions of Echinococcosis multilocularis.

[0006] The present invention also provides a drug for treating and / or preventing multilocular echinococcosis, which contains (or its active ingredient is) Apoptozole.

[0007] The present invention also provides a drug for reducing the infection or metastasis of Echinococcus multilocularis, which contains (or its active ingredient is) Apoptozole.

[0008] The present invention also provides a drug for inhibiting the development of Echinococcus multilocularis, which contains (or its active ingredient is) Apoptozole.

[0009] The present invention also provides a drug for killing Echinococcus multilocularis, which contains (or its active ingredient is) Apoptozole.

[0010] The present invention also provides a method for reducing multilocular echinococcosis lesions, which contains (or its active ingredient is) Apoptozole.

[0011] The development of Echinococcus multilocularis may refer to the development of various stages thereof, such as the development of the protoscolex.

[0012] The killing of Echinococcus multilocularis can be killing at various stages, such as killing the protoscolecere or the tapeworm stage vesicles. The killing of Echinococcus multilocularis can be destroying its tissue structure.

[0013] The present invention finds that Apoptozole can treat and / or prevent multilocular echinococcosis, and has a killing effect on both the protoscolecere and the tapetum stage vesicle stage of multilocular echinococcosis larvae in vitro, can destroy the protoscolecere hook, sucker, microvilli and other tissue structures, cause the tapetum stage vesicle collapse, and can also significantly inhibit the development of protoscolecere to cysts in the secondary infection model of multilocular echinococcosis mice, and the infection rate and liver metastasis rate are both reduced. It shows that Apoptozole can be used as a drug for the treatment of multilocular echinococcosis.

[0014] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 :The killing effect of different concentrations of Apoptozole on the protoscolex of Echinococcus multilocularis (methylene blue staining).

[0016] Figure 2 :Effects of different concentrations of Apoptozole intervention on the survival rate of protoscolex of Echinococcus multilocularis.

[0017] Figure 3 :Effects of Apoptozole (20μM) intervention for 12 days on the protoscolex activity of Echinococcus multilocularis.

[0018] Figure 4 :Effects of Apoptozole (20μM) on the ultrastructure of the protoscoleme of Echinococcus multilocularis after 12-day intervention.

[0019] Figure 5 :The effect of apoptozole (20 μM) on the vesicle activity of the tapeworm stage of Echinococcus multilocularis for 12 days. Bar value = 750 μm.

[0020] Figure 6 :The effect of apoptozole (20 μM) intervention of Echinococcus multilocularis protoscolex on infection and parasitism in mice for 12 days. A: Gross observation; B: Gross observation of lesions; C: Infection rate, the number on the left of “ / ” indicates the number of infected mice, the number on the right of “ / ” indicates the total number of mice in this group; D: Liver metastasis rate, the number on the left of “ / ” indicates the number of mice with liver metastasis, the number on the right of “ / ” indicates the total number of mice in this group; E: Lesion weight. DETAILED DESCRIPTION

[0021] The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources. The quantitative tests in the following examples are all set up for at least three replicates. The following examples use GraphPad Prism8.3.0 statistical software to process the data.

[0022] Apoptozole: Abcam product; Catalog number: ab287038; CAS: 1054543-47-3.

[0023] Example 1: Apoptozole can treat multilocular echinococcosis This example found that Apoptozole can treat multilocular echinococcosis, and the test is as follows: 1. In vitro culture of Echinococcus multilocularis protoscoleces for Apoptozole intervention: Take a breeding mouse with abdominal infection of Echinococcus multilocularis for more than 6 months, kill it by cervical dislocation, and completely immerse it in 75% ethanol aqueous solution, and sterilize the body surface for 1 minute. Separate the lesion tissue of abdominal infection under sterile conditions, wash the residual blood with sterile PBS, and place it in a sterilized 100-mesh steel cell sieve (steel mesh), put the steel mesh in a plate containing PBS solution, cut the lesion tissue into pieces, and grind it on the steel mesh with the piston handle of a 5 ml (mL) syringe. Add PBS solution to the steel mesh, stir the ground lesion tissue continuously, and filter and collect the protoscoleces of Echinococcus multilocularis.

[0024] The protoscole (i.e., larval stage) of Echinococcus multilocularis were isolated under aseptic conditions in vitro and placed in a 96-well plate containing 200 μL of culture medium (1% penicillin-streptomycin + 10% (volume percentage) fetal bovine serum + RPMI-1640 culture medium), and then different concentrations of Apoptozole were added to the culture system for intervention (Apoptozole was dissolved in DMSO and prepared into a 20 mM stock solution). The experiment set up a solvent control group (the final concentration was 0.1% DMSO control group, that is, the same volume of DMSO as the intervention group was added to the culture system, and the final concentration was 0.1%), an Apoptozole intervention group (the concentration of Apoptozole was set to 1 μM, 5 μM, 10 μM, and 20 μM, that is, the final concentrations of Apoptozole in the culture system were 1 μM, 5 μM, 10 μM, and 20 μM, respectively). The protoscolex were collected 3, 6, 9 and 12 days after intervention, and the activity of protoscolex was detected by methylene blue staining. The morphology and ultrastructure changes of the insects were observed using optical microscopy and scanning electron microscopy.

[0025] The experimental results are shown in Figure 1 and Figure 2 After 12 days of treatment with Apoptozole (1 μM, 5 μM, 10 μM, 20 μM), the survival rates of the protoscolecere decreased to 78 ± 1.25%, 75 ± 2.72%, 59 ± 4.39%, and 0.7 ± 0.30%, respectively. At this time, the survival rate of the protoscolecere in the solvent control group was 82 ± 3.31%. Optical microscopy showed that after 12 days of Apoptozole (20 μM) intervention, the protoscolecere shrank and turned black, the head hook fell off, and the worm body lysed and died; while the protoscolecere in the solvent control group had intact morphology and structure, good vitality, and some protoscolecere segments began to develop into cysts ( Figure 3 Scanning electron microscopy also found that after 12 days of Apoptozole (20 μM) intervention, the protoscolex morphology was wrinkled, the head hook fell off, the sucker structure was destroyed, and the microvilli were truncated ( Figure 4 ). This indicates that Apoptozole (20 μM) has a good killing effect on the protoscoleces of Echinococcus multilocularis cultured in vitro.

[0026] 2. In vitro culture of Echinococcus multilocularis tapeworm vesicles for Apoptozole intervention: Vesicles of Echinococcus multilocularis were cultured under sterile conditions in vitro from the protoscolecus to the tapetum stage. Microvesicles with a diameter of 2-3 mm were selected and placed in a 24-well plate containing 500 μL of culture medium (1% penicillin-streptomycin + 10% fetal bovine serum + RPMI-1640) for culture. Apoptozole (20 μM, i.e., the final concentration of Apoptozole in the culture system was 20 μM) was added for intervention (i.e., Apoptozole intervention group). A solvent control group (DMSO of the same volume as the intervention group was added to the culture system, with a final concentration of 0.1%) was set up. Microscopic observation showed that after 3 days of Apoptozole (20 μM) intervention, the vesicles began to collapse and the vesicle wall lost its luster; after 12 days of Apoptozole (20 μM) intervention, the vesicle wall collapsed severely and the germinal layer fell off, while the vesicle structure of the solvent control group was intact ( Figure 5 ). This indicates that Apoptozole has a good killing effect on the tapeworm vesicles of Echinococcus multilocularis cultured in vitro.

[0027] 3. Evaluation of pathogenicity of Echinococcus multilocularis protoscolex cultured in vitro in mice after intervention with Apoptozole: The protoscoleces of Echinococcus multilocularis were isolated in vitro and cultured in 6-well plates containing 4 mL of culture medium (1% penicillin-streptomycin + 10% fetal bovine serum + RPMI-1640). They were divided into a solvent control group (final concentration of 0.1% DMSO control group, i.e., DMSO of the same volume as the intervention group was added to the culture system, with a final concentration of 0.1%) and an Apoptozole intervention group (20 μM, i.e., the final concentration of Apoptozole in the culture system was 20 μM), and cultured in vitro for 28 days; then the two groups of protoscoleces were intraperitoneally injected into female Balb / c mice at a dose of 5000 per mouse, with 8 mice in each group. Mice and lesion samples were collected 24 weeks after protoscolece inoculation to detect and compare the ability of protoscoleces to develop into tapetal vesicles and the weight of lesions, and the infection rate and metastasis rate were calculated.

[0028] Infection rate = number of infected mice / total number of mice in the group; Metastasis rate = number of mice with liver metastasis / total number of mice in the group.

[0029] Female Balb / c mice, 6 to 7 weeks old, weighing 18 to 22 g, were used in the experiment. Be careful to operate steadily, otherwise the injury will be serious; when injecting the protoscoleces of multilocular Echinococcus, make sure that no air enters.

[0030] The experimental results are shown in Figure 6 , 24 weeks after inoculation, the infection rate of mice in the solvent control group was 85.7%, and peritoneal cysts and liver metastatic lesions were observed, with a metastasis rate of 71.5%; the infection rate of mice in the Apoptozole intervention group (20μM) was 25%, and the metastasis rate was 0%, which was significantly lower than that in the solvent control group. The lesion weight of the Apoptozole intervention group (2.125mg) was significantly lower than that of the solvent control group (3712.5mg). This indicates that Apoptozole can be used as a drug for the treatment or prevention of multilocular echinococcosis.

[0031] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Use of apoptozole in the preparation of a drug for treating and / or preventing multilocular echinococcosis; or, Use of Apoptozole in the treatment and / or prevention of multilocular echinococcosis.

2. Use of Apoptozole in the preparation of drugs for reducing infection or metastasis of Echinococcus multilocularis; Or, Use of Apoptozole in reducing infection or metastasis of Echinococcus multilocularis.

3. Application of Apoptozole in the preparation of products for inhibiting the development of Echinococcus multilocularis; Or, Use of apoptozole in inhibiting the development of Echinococcus multilocularis.

4. Application of Apoptozole in the preparation of products for killing Echinococcus multilocularis; Or, Use of Apoptozole in killing Echinococcus multilocularis.

5. Application of Apoptozole in the preparation of products for reducing multilocular echinococcosis lesions; Or, Apoptozole for reducing lesions of multilocular echinococcosis.

6. Medications for the treatment and / or prevention of multilocular echinococcosis containing Apoptozole.

7. Drugs for reducing infection or metastasis of Echinococcus multilocularis containing Apoptozole.

8. A drug for inhibiting the development of Echinococcus multilocularis containing Apoptozole.

9. Drugs used to kill Echinococcus multilocularis tapeworms, containing Apoptozole.

10. Used to reduce the lesions of multilocular echinococcosis, containing Apoptozole.

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