Use of aoh1996 in the preparation of a medicament for treating echinococcosis

Abstract

The application provides application of AOH1996 in preparation of a drug for treating echinococcosis, and belongs to the technical field of biological medicine. The application of AOH1996 in preparation of the drug for treating echinococcosis. Experiments prove that AOH1996 has a killing effect on in-vitro hydatid cystic echinococcus protocercous and vesicles. Therefore, the technical scheme provided by the application provides technical support for efficiently preventing and treating hydatid disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of AOH1996 in the preparation of drugs for treating echinococcosis. Background Technology

[0002] Echinococcosis, also known as hydatid disease, is a serious zoonotic parasitic disease caused by the larvae of the Echinococcus tapeworm, which severely endangers human health and livestock development. The disease is distributed worldwide, and its lesions are usually located in the liver, but can also develop in the lungs. Currently, the main treatment for hydatid disease relies on surgery, specifically surgical removal of the tumor-like cysts from the liver.

[0003] In recent years, surgical resection has offered promising prospects for the cure of echinococcosis, effectively reducing the recurrence rate. However, surgical treatment is only suitable for early-stage lesions, at which point the lesion can be completely removed without affecting surrounding organs. But since most echinococcosis patients are diagnosed at an advanced stage, the cysts are often large and located near vital organs, making complete radical removal impossible. Furthermore, during surgery, the scolex may be missed or remain, requiring adjunctive drug treatment to prevent recurrence.

[0004] AOH1996 is a small-molecule proliferating cell nuclear antigen (PCNA) inhibitor with oral activity. It is obtained by adding a methoxy group to the meta-position of the terminal benzene ring of AOH1160, which enhances the interaction between PCNA and RPB1, the largest subunit of RNA polymerase II. Existing technology reports that AOH1996 can selectively kill cancer cells and is often used in the preparation of anticancer drugs. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a new use for AOH1996, namely, the use of AOH1996 in the preparation of drugs for treating echinococcosis.

[0006] This invention provides the application of AOH1996 in the preparation of drugs for treating echinococcosis.

[0007] Preferably, the echinococcosis includes multilocular echinococcosis.

[0008] Preferably, the drug comprises at least one of the following: inhibiting the activity of Echinococcus multilocularis prostaglandins and / or Echinococcus multilocularis vesicles, disrupting the morphology of Echinococcus multilocularis prostaglandins and / or Echinococcus multilocularis vesicles, and promoting the death of Echinococcus multilocularis prostaglandins and / or Echinococcus multilocularis vesicles.

[0009] Preferably, the dosage form of the drug includes an oral dosage form;

[0010] The oral dosage form includes at least one of the following: tablets, capsules, and powders.

[0011] Preferably, the concentration of AOH1996 in the drug is not less than 20 μM.

[0012] Preferably, the concentration of AOH1996 in the drug is 40–80 μM.

[0013] This invention provides the application of AOH1996 in the preparation of formulations that kill Echinococcus larvae in vitro.

[0014] Preferably, the echinococcosis comprises the echinococcosis prostomy segment and / or echinococcosis cyst.

[0015] Preferably, the echinococcosis includes Echinococcus multilocularis.

[0016] This invention provides a method for killing Echinococcus larvae in vitro, wherein a sample containing Echinococcus larvae is cultured in an environment containing AOH1996; the concentration of AOH1996 is not less than 20 μM.

[0017] This invention provides the application of AOH1996 in the preparation of drugs for treating echinococcosis. This invention investigated the efficacy of AOH1996 against echinococcosis using different life-stage morphologies of echinococcosis (protoscolex and cysts). Experimental results showed that AOH1996 effectively inhibited the activity of the protoscolex and cysts of *Echinococcus multilocularis*. Administration of AOH1996 disrupted the original morphology of the protoscolex and cysts, promoting the death of the protoscolex and achieving a dose-dependent killing effect on echinococcosis. Therefore, the application scheme provided by this invention will provide technical support for the efficient prevention and control of echinococcosis. Attached Figure Description

[0018] Figure 1 The graph shows the results of protoscolex viability determination after in vitro culture of different concentrations of AOH1996 for 96 hours.

[0019] Figure 2 Figure showing the mortality rate of different concentrations of AOH1996 on the protoscolex of Echinococcus multilocularis;

[0020] Figure 3 The graph shows the cytotoxic effect of AOH1996 on in vitro vesicles.

[0021] Figure 4 The diagram shows the changes in the internal structure of the protoscolex after 5 days of treatment with AOH1996. Detailed Implementation

[0022] This invention provides the application of AOH1996 in the preparation of drugs for treating echinococcosis.

[0023] In this invention, the molecular formula of AOH1996 is C26 H 22 N2O4, CAS: 2089314-64-5, structural formula is shown in Formula I:

[0024]

[0025] In this invention, the echinococcosis preferably includes multilocular echinococcosis (alveolar echinococcosis). The echinococcosis includes echinococcosis in animals or humans. The echinococcosis is preferably hepatic echinococcosis, depending on the affected site.

[0026] In this invention, the dosage form of the drug preferably includes an oral dosage form. The oral dosage form preferably includes at least one of the following: tablets, capsules, and powders. The oral dosage form is preferably prepared in combination with pharmaceutically acceptable excipients, depending on the dosage form. This invention does not limit the preparation method of the oral dosage form; any method well-known in the art can be used. The concentration of AOH1996 in the drug is preferably not less than 20 μM, more preferably 40–80 μM. The drug is used continuously for at least 5 days, more preferably 6–10 days. The dosage of the drug is preferably 40–80 μM.

[0027] In this invention, the drug preferably includes at least one of the following: inhibiting the activity of the prostomium and / or vesicles of Echinococcus multilocularis, destroying the morphology of the prostomium and / or vesicles of Echinococcus multilocularis, and promoting the death of the prostomium and / or vesicles of Echinococcus multilocularis.

[0028] In one embodiment of the present invention, the effects of different concentrations of AOH1996 on the activity and mortality of protoscolex of Echinococcus multilocularis were evaluated. PI staining results showed that, compared with the blank control group and the DMSO group, the number of live protoscolex was significantly reduced at a drug concentration of 20 μM, while the number of dead protoscolex was significantly increased at drug concentrations of 40 μM and 80 μM.

[0029] In another embodiment of the invention, the effects of different concentrations of AOH1996 on the segment morphology of *Echinococcus multilocularis* were evaluated. The results showed that the protoscolex in the control group and the DMSO group had intact cyst walls containing vesicles of varying sizes and shapes, arranged regularly, and containing various cell types. After treatment with AOH1996, the protoscolex shrank, became loosely structured, incomplete in morphology, with thinner cyst walls and a reduced number of cilia. This indicates that administration of AOH1996 can affect the normal morphology of *Echinococcus multilocularis* segments, thereby leading to their normal growth.

[0030] In another embodiment of the present invention, the effects of different concentrations of AOH1996 on the morphology of Echinococcus multilocularis vesicles were evaluated. The results showed that the vesicles in the DMSO group were plump and smooth, and no collapse was observed. However, the vesicles treated with AOH1996 showed significant changes in morphology, with collapse and shrinkage, affecting the normal morphology of the vesicles.

[0031] This invention provides the application of AOH1996 in the preparation of formulations that kill Echinococcus larvae in vitro.

[0032] In this invention, the echinococcosis preferably comprises the prostomium and / or cysts of the echinococcosis. The echinococcosis preferably comprises multilocular echinococcosis. This invention does not impose any particular limitation on the type of formulation; any formulation type well-known in the art may be used.

[0033] This invention provides a method for killing Echinococcus larvae in vitro, wherein a sample containing Echinococcus larvae is cultured in an environment containing AOH1996; the concentration of AOH1996 is not less than 20 μM.

[0034] In this invention, the concentration of AOH1996 is preferably 40–80 μM, and most preferably 60 μM. The environment containing AOH1996 is preferably a culture medium containing AOH1996. The culture medium is preferably DMEM medium free of fetal bovine serum. The culture time is preferably 20–26 h, more preferably 24 h.

[0035] In this embodiment of the invention, AOH1996 was used to kill Echinococcus multilocularis larvae. The results showed that 20 μM AOH1996 significantly reduced the activity of the vesicles compared with the blank group and the DMSO control group. Furthermore, the activity curve showed that the inhibition rate of the protoscolex increased with the increase of drug concentration and the duration of treatment. This effect was dose-dependent and time-dependent. After 7 days of treatment with 80 μM AOH1996, the vesicle mortality rate reached 95%.

[0036] The following examples illustrate the application of AOH1996 provided by the present invention in the preparation of drugs for treating echinococcosis, but these examples should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1

[0038] The effect of AOH1996 on the prostomium of multilocular echinococcosis in vitro

[0039] After euthanizing gerbils infected with *Echinococcus multilocularis* by cervical dislocation, the gerbils were sterilized by spraying their surface thoroughly with 75% alcohol and transferred to a laminar flow hood for aseptic isolation of the protoscolex. The *Echinococcus multilocularis* cysts were detached from the gerbil tissue, and any remaining tissue was removed. The cysts were then placed in pre-cooled PBS buffer (containing 100 U / mL penicillin and 100 U / mL streptomycin) and washed three times. The cyst tissue was then minced using ophthalmic scissors, and the mixture was transferred to a beaker with an 80-mesh copper mesh. The mixture was slowly ground with a pestle until all the contents were filtered through the 80-mesh copper mesh. An appropriate amount of PBS buffer was added, allowing the worms to settle naturally. The supernatant was discarded until the solution was clear. Under a microscope, the protoscolex worms were observed to have a plump, oval structure with clear and intact features and obvious peristalsis. The protostomia were aliquoted into DMEM medium (100 U / mL penicillin, 100 U / mL streptomycin) without fetal bovine serum and cultured in a 37°C, 5% CO2 incubator.

[0040] Experimental groups: blank group, DMSO group (solvent control), 20 μM AOH 1996 group, 40 μM AOH 1996 group, and 80 μM AOH 1996 group. 48-well plates were used, with 1 mL of the drug in each well. Approximately 1000 protoscolex were added to each group's culture medium. The plates were incubated at 37°C in a 5% CO2 incubator. The effects of the drug on the morphology and viability of *Echinococcus multilocularis* protoscolex were observed under a microscope at different incubation times.

[0041] Data analysis method: Starting 24 hours after drug administration, 100 μL of culture medium (containing an average of 90–120 protoscolex) was uniformly drawn from each well daily, stained with 1% PI dye for 15 min, and then washed off with PBS buffer. The viability of the protoscolex was observed under a fluorescence microscope. Live protoscolex remained unstained and showed activity; protoscolex that stained with strong red fluorescence and showed no obvious peristalsis were considered dead. The experiment was repeated three times. The protoscolex mortality rate was calculated according to Formula I.

[0042] Mortality rate of protoceratops (%) = Number of protoceratops that died / 100% Formula I

[0043] See results Figure 1 After staining with 1% PI, compared with the blank control group and the DMSO group, the number of dead protoscolex was significantly increased at drug concentrations of 40 μM and 80 μM.

[0044] Mortality results are shown Figure 2 Compared with the blank group and the DMSO control group, the vesicle inhibition rate of 20 μMAOH1996 was significantly reduced. The activity curve showed that the inhibition rate of protoscolex increased with the increase of drug concentration and the duration of treatment. This effect was dose-dependent and time-dependent. After 7 days of treatment with 80 μMAOH1996, the vesicle mortality rate reached about 95%.

[0045] Example 2

[0046] The effect of AOH1996 on multilocular echinococcosis vesicles in vitro

[0047] The processed protostomes (approximately 2000 per mL) were transferred to DMEM medium containing 10% fetal bovine serum (100 U / mL penicillin, 100 U / mL streptomycin), and 1×10⁻⁶ mol / mL of the medium was added. 6 Rat hepatocellular carcinoma cells (RH35) were co-cultured with protostomes in a 37°C, 5% CO2 incubator for 1–2 months, with half the medium replaced every 3 days. Vesicles with a diameter of approximately 2–4 mm were selected for experiments.

[0048] Experimental groups: blank group, DMSO group (solvent control), 20 μM AOH 1996 group, 40 μM AOH 1996 group, and 80 μM AOH 1996 group. Vesicles were transferred to PBS and washed three times, then aliquoted into 24-well plates (20–30 vesicles per well), with 2 mL of medium per well. DMEM medium (100 U / mL penicillin and 100 U / mL streptomycin) without fetal bovine serum was added, and the plates were incubated at 37°C in a 5% CO2 incubator. Data analysis: Vesicle morphology was observed under an inverted microscope starting 24 hours after drug administration.

[0049] See results Figure 3 Under an inverted microscope, the vesicles in the DMSO group were full and smooth, with no collapse observed. Compared to the DMSO group, after 9 days of treatment with different concentrations of AOH1996, the vesicle morphology changed significantly, with collapse and contraction observed.

[0050] Example 3

[0051] AOH1996 Effects on Changes in the Internal Structure of the Protoscolex

[0052] While measuring viability, corresponding protoscolex sections were randomly selected for sectioning to observe changes in their internal structure. The tissue was washed three times with PBS, allowed to settle naturally, and fixed with 4% paraformaldehyde for 24 hours. The supernatant was removed, and the tissue was dehydrated and impregnated with paraffin in a series of alcohol solutions: 75% ethanol for 4 hours, 85% ethanol for 2 hours, 90% ethanol for 2 hours, 95% ethanol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, and paraffin I, II, and III at 65°C for 1 hour. The paraffin-impregnated tissue was then embedded in an embedding machine, fixed, and trimmed. The trimmed paraffin blocks were sectioned using a paraffin microtome to a thickness of 4 μm and observed under a scanning electron microscope.

[0053] Figure 4The internal structural changes of the protoscolex after 5 days of treatment with AOH1996 were observed. Electron microscopy revealed that the protoscolex in the control and DMSO groups had intact vesicle walls containing vesicles of varying sizes and shapes, arranged regularly, and containing various cell types. After treatment with AOH1996, the protoscolex showed shrinkage, loosening of structure, incomplete morphology, thinning of vesicle walls, and a decrease in the number of cilia.

[0054] The results of the above examples demonstrate that AOH1996 has a killing effect on the protoscolex and vesicles of Echinococcus multilocularis in vitro. These findings provide technical support for the efficient prevention and control of echinococcosis.

[0055] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of AOH1996 in the preparation of a drug for treating echinococcosis.

2. Use according to claim 1, characterized in that, The echinococcosis includes multilocular echinococcosis.

3. Use according to claim 1, characterized in that, The drug is used for at least one of inhibiting the activity of multilocular echinococcosis protoscolex and / or multilocular echinococcosis vesicle, destroying the morphology of multilocular echinococcosis protoscolex and / or multilocular echinococcosis vesicle, and promoting the death of multilocular echinococcosis protoscolex and / or multilocular echinococcosis vesicle.

4. The use according to claim 1, characterized in that, The dosage form of the drug includes an oral dosage form. The oral dosage form includes at least one of a tablet, a capsule, and a powder.

5. The use according to claim 1, characterized in that, The concentration of AOH1996 in the drug is not less than 20 μM.

6. The use according to claim 1, characterized in that, The concentration of AOH1996 in the drug is 40-80 μM.

7. Use of AOH1996 in the preparation of a preparation for killing echinococcosis in vitro.

8. Use according to claim 7, characterized in that, The echinococcosis includes echinococcosis protoscolex and / or echinococcosis cyst.

9. Use according to claim 7 or 8, characterized in that, The echinococcosis includes multilocular echinococcosis.

10. A method for killing Echinococcus outside of a disease diagnostic purpose and a disease therapeutic purpose, characterized by, An echinococcosis-containing sample is cultured in an environment containing AOH1996. The concentration of AOH1996 is not less than 20 μM.

Images