Use of a small molecule inhibitor thonningianin a in the preparation of a drug for treating multilocular hydatidosis

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a small molecule inhibitor Thonningianin A in preparation of a drug for treating multilocular hydatid disease. 50 The application takes EmAGO2 protein which is specifically highly expressed by germinal layer cells of multilocular hydatid as a target, and obtains a small molecule inhibitor Thonningianin A, the binding affinity constant KD of which to EmAGO2 is 101.3 nM, and the equilibrium dissociation constant KD is 5.51 nM. In vitro experiments show that the IC 9.22 μM, and can significantly inhibit cyst growth; in vivo experiments show that the 40 mg / kg oral treatment effect is equal to that of albendazole which is a first-line drug, and the safety is good. The application provides a new target and a candidate drug for the treatment of multilocular hydatid disease, and is expected to improve the clinical difficulties of limited treatment effect and easy drug resistance of an existing treatment scheme.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a small molecule inhibitor, Thonningianin A, in the preparation of drugs for treating echinococcosis. Background Technology

[0002] Alveolar echinococcosis (AE), also known as alveolar echinococcosis, is a serious zoonotic parasitic disease caused by infection with the larvae (alveolar cysts) of the tapeworm *Echinococcus multilocularis*. The typical liver lesions present as irregularly shaped white clusters or masses of small vesicles, with indistinct boundaries between the lesions and the host liver tissue, exhibiting infiltrative and exophytic growth. Because the lesions can progress in a tumor-like, invasive manner and metastasize to distant organs, AE is clinically referred to as "parasitic cancer." This disease is characterized by its severe severity and prolonged course. Clinical data shows that the mortality rate for AE patients who do not receive standardized or adequate treatment can reach approximately 90% within 10 to 15 years of onset, seriously threatening the lives and health of patients and placing a heavy burden on patients' families and the public healthcare system.

[0003] Current clinical treatment strategies for echinococcosis primarily rely on surgical resection, supplemented by adjuvant drug therapy. However, these strategies face numerous insurmountable bottlenecks in practical clinical application. Surgically, most patients exhibit multifocal lesions with diffuse infiltrative growth within the liver tissue, meaning only about 30% of patients meet the criteria for surgical resection. Even when surgery is possible, the recurrence rate remains high, making overall treatment outcomes difficult to achieve. Drug-wise, albendazole is currently the only approved drug for echinococcosis treatment. However, this class of drugs suffers from limited overall efficacy and a tendency to develop drug resistance with long-term use, further exacerbating the clinical challenges of echinococcosis treatment. Therefore, developing novel, highly effective, and low-toxicity drugs for echinococcosis with entirely new mechanisms of action has become a critical medical challenge that urgently needs to be addressed.

[0004] From the perspective of the parasite's biological mechanisms, *Echinococcus multilocularis* larvae can proliferate indefinitely in the human body in a tumor-like manner. The core driving mechanism lies in the continuous proliferation and differentiation of germinal layer cells, which continuously form new vesicles and generate new protoscolex, promoting the invasive growth and spread of lesions. In recent years, research on the maintenance and regulation mechanisms of parasite stem cells has provided new research directions for the discovery of targets for anti-*Echinococcus multilocularis* drugs. However, most of the reported anti-*Echinococcus multilocularis* drug targets have high sequence homology with their corresponding homologous proteins in the host, making it easy for these drugs to have off-target effects on normal host proteins. Furthermore, existing target drugs have very limited inhibitory effects on the germinal layer stem cells that drive the unlimited proliferation of lesions, making it difficult to fundamentally block the proliferation and invasion of lesions. Therefore, developing a novel drug target that can effectively inhibit the proliferative activity of *Echinococcus multilocularis* larvae while possessing high host specificity and low systemic toxicity remains a core technical problem that urgently needs to be solved in the treatment of *Echinococcus multilocularis*.

[0005] Based on whole-genome alignment and single-cell transcriptome sequencing analysis, it was discovered that *Echinococcus multilocularis* has lost the Piwi gene family, essential for maintaining traditional stem cell pluripotency, during its evolutionary process. Instead, it has evolved a new group of Argonaute proteins unique to this tapeworm species, named the EmAGOs family. This family includes three functional members: EmAGO2, EmAGO3, and EmAGO4. EmAGO2 exhibits highly specific expression in the germinal layer cells of *Echinococcus multilocularis* larvae. This expression pattern suggests that EmAGO2 may play a crucial role in the lineage maintenance and proliferation regulation of *Echinococcus multilocularis* stem cells, and possesses potential value as a drug target for combating *Echinococcus multilocularis* disease. Further amino acid sequence alignment analysis showed that EmAGO2 has low amino acid homology with human and mouse AGO2 proteins, and there are significant differences in their key functional domains. Based on this species difference at the sequence and structural level, drugs targeting EmAGO2 protein are less likely to interfere with the normal physiological function of the host's own AGO2 protein. The risk of off-target toxicity can be reduced from the target design level, and theoretically, the drugs have lower systemic toxicity and a wider therapeutic window, which can provide a guarantee for the safety of clinical drug use.

[0006] To date, there are no existing research reports on screening small molecule inhibitors using the EmAGO2 protein, a specific protein of Echinococcus multilocularis, as a direct drug target, and on using the screened small molecule inhibitors for the treatment of Echinococcus multilocularis larvae. Summary of the Invention

[0007] The purpose of this invention is to provide the application of the small molecule inhibitor Thonningianin A in the preparation of drugs for treating echinococcosis, providing reliable technical support for the efficient prevention and treatment of echinococcosis, and laying a solid experimental foundation for the clinical application of Thonningianin A in the treatment of echinococcosis.

[0008] The objective of this invention is achieved through the following technical solution: This invention provides the use of Thonningianin A in the preparation of a drug for treating echinococcosis, wherein the structural formula of Thonningianin A is shown below: .

[0009] Furthermore, the Thonningianin A targets the EmAGO2 protein and binds to it with high affinity.

[0010] Furthermore, the binding affinity constant KD of Thonningianin A to EmAGO2 protein is 101.3 nM, and the equilibrium dissociation constant KD is 5.51 nM.

[0011] Furthermore, the aforementioned echinococcosis is caused by infection with the vesicular larvae of Echinococcus multilocularis. Its typical liver lesions are irregularly shaped white vesicle clusters or masses formed by the aggregation of a large number of small vesicles, which are not clearly demarcated from the host liver tissue and exhibit infiltrative and exophytic growth.

[0012] Furthermore, the drug is in the form of an oral dosage form.

[0013] Furthermore, the Thonningianin A is dissolved in DMSO and diluted with physiological saline before administration, and the dosage of Thonningianin A is not less than 40 mg / kg.

[0014] Furthermore, when administered orally at a dose of 40 mg / kg, Thonningianin A reduced the lesion area and significantly decreased the number and weight of echinococcosis cysts in individuals infected with multilocular echinococcosis, achieving a therapeutic effect comparable to that of albendazole at 40 mg / kg.

[0015] The present invention also provides a pharmaceutical composition for treating echinococcosis, comprising an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient is Thonningianin A or a pharmaceutically acceptable salt thereof or a hydrate thereof.

[0016] Furthermore, the dosage form of the pharmaceutical composition is an oral dosage form.

[0017] Furthermore, the oral dosage form is selected from one of tablets, granules, powders, capsules, liposome-loaded drugs, oral liquids, or lozenges.

[0018] The beneficial effects of this invention are as follows: The EmAGO2 protein targeted by this invention is a specific protein of Echinococcus multilocularis. Its amino acid sequence and key functional domains have low homology with human and mouse AGO2 proteins, especially in the substrate binding domain and the interaction region with cofactors. Drugs developed based on this target are less likely to interfere with the normal physiological function of the host's own AGO protein. The off-target toxicity risk of the drug is reduced from the target design level. Theoretically, it has lower systemic toxicity and a wider therapeutic window, which can effectively ensure the safety of clinical drug use.

[0019] This invention clarifies for the first time the application value of Thonningianin A, a small molecule inhibitor targeting the EmAGO2 protein of Echinococcus multilocularis, in the treatment of Echinococcus multilocularis cysts. It fills the gap in the existing technology for developing anti-Echinococcus multilocularis cyst drugs with EmAGO2 as a direct drug target, and provides a new technical path for the treatment of this disease. It breaks through the technical bottlenecks of existing treatments, such as high target homology, high off-target risk, and limited inhibitory effect on germinal stem cells that drive the unlimited proliferation of lesions.

[0020] This invention screened for Thonningianin A using computer-aided drug design technology, and independently verified by both micro-thermophoresis and surface plasmon resonance techniques, confirming that Thonningianin A binds to EmAGO2 protein with a significant high affinity. The binding affinity constant KD is 101.3 nM, and the equilibrium dissociation constant KD is 5.51 nM. This clearly elucidates the molecular mechanism by which Thonningianin A exerts its anti-hydatid effect, providing a solid theoretical basis for the subsequent drug development and clinical translation of this compound.

[0021] In vitro experiments showed that Thonningianin A has a strong inhibitory activity against the protoscolex of Echinococcus multilocularis. The survival rate of the protoscolex decreased significantly in a dose-dependent manner with increasing drug concentration, and its half-maximal inhibitory concentration (IC50) for the protoscolex was 9.22 μM. At the same time, this compound can significantly inhibit the growth and development of new vesicles formed by the differentiation of protoscolex. The treated vesicles exhibited a distinct shrunken phenotype, and the degree of shrunkenness increased significantly with increasing drug concentration, thus achieving effective inhibition of different developmental stages of Echinococcus multilocularis.

[0022] In vivo animal experiments showed that after oral administration of 40 mg / kg Thonningianin A every 3 days for 1 month, the lesion area of ​​Echinococcus multilocularis infected mice was significantly reduced, and the number and weight of Echinococcus cysts were significantly decreased. The therapeutic effect was comparable to that of albendazole, a first-line clinical drug. There was no significant difference in the body weight of mice in different groups during the treatment period, confirming that Thonningianin A has good in vivo safety at effective therapeutic doses.

[0023] The application scheme of Thonningianin A in the preparation of drugs for treating echinococcosis provided by this invention provides reliable technical support for the efficient prevention and treatment of echinococcosis, lays a solid experimental foundation for the application of Thonningianin A in the clinical treatment of echinococcosis, and is expected to improve the current clinical dilemma of limited drugs for the treatment of echinococcosis, poor efficacy, and easy development of drug resistance with long-term use. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This image shows the results of immunofluorescence staining to detect the expression and localization of EmAGO2 protein in Echinococcus multilocularis tissue, illustrating the expression and distribution characteristics of EmAGO2 protein in the germinal layer cells of Echinococcus multilocularis.

[0026] Figure 2 This graph shows the results of micro-thermophoresis (MST) to detect the binding affinity of the small molecule inhibitor Thonningianin A to EmAGO2 protein. The horizontal axis represents the ligand concentration, and the vertical axis represents the change in fluorescence signal response. This was used to determine the binding affinity constant KD between the two proteins.

[0027] Figure 3 The image shows the results of surface plasmon resonance (SPR) technology used to verify the specific binding of the compound to the EmAGO2 protein; in which... Figure 3 A represents the binding response curves of the DMSO solvent control group to different concentrations of EmAGO2 protein. Figure 3 B shows the specific binding response curves of Thonningianin A with different concentrations of EmAGO2 protein, used to verify binding specificity and determine the equilibrium dissociation constant KD.

[0028] Figure 4The figure shows the survival rate of protoscars of Echinococcus multilocularis after in vitro treatment with different concentrations of Thonningianin A for 48 hours. Figure 4 Image A shows the morphological observation and PI staining fluorescence detection of Echinococcus multilocularis protoscars after treatment with different concentrations of Thonningianin A for 48 hours. Figure 4 Figure B is a bar chart showing the survival rate of Echinococcus multilocularis protocercariae after treatment with different concentrations of Thonningianin A for 48 hours. Figure 4 C represents the dose-response curve of the inhibitory effect of Thonningianin A on the protocercariae of Echinococcus multilocularis.

[0029] Figure 5 The figures show the morphological changes and vesicle viability of vesicles differentiated from protocercariae of Echinococcus multilocularis after in vitro treatment with different concentrations of Thonningianin A for 48 h, illustrating the inhibitory effect of Thonningianin A on vesicle growth and development and its dose-dependent effect.

[0030] Figure 6 The graph shows the weight changes in mice infected with Echinococcus multilocularis during 30 days of continuous treatment with the Thonningianin A small molecule inhibitor, used to evaluate the in vivo safety of Thonningianin A at a dose of 40 mg / kg.

[0031] Figure 7 The following are the results of liver morphology observation and cyst weight statistics in mice infected with Echinococcus multilocularis after 30 days of treatment with a Thonningianin A small molecule inhibitor; including gross morphological images of the livers of mice in each group. Figure 7 (A) Liver weight statistics chart ( Figure 7 Statistical chart of weight of Echinococcus larvae (B) and Echinococcus cysts (B) Figure 7 (C) was used to visually evaluate the therapeutic effect of Thonningianin A against Echinococcus multilocularis in vivo. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] Example 1: Validation of the activity of Thonningianin A targeting EmAGO2 in the treatment of multilocular echinococcosis. 1. Validation of target expression localization of EmAGO2 protein Immunofluorescence staining was used to detect the expression and distribution characteristics of EmAGO2 protein in Echinococcus multilocularis larvae. The results are as follows: Figure 1 As shown, immunofluorescence staining results confirmed that EmAGO2 protein was specifically highly expressed in the germinal layer cells of Echinococcus multilocularis, suggesting that EmAGO2 may play a key regulatory role in the maintenance and proliferation of Echinococcus multilocularis stem cell lineages and has the potential to serve as a drug target for anti-Echinococcus multilocularis disease.

[0038] 2 Virtual screening of small molecule compounds targeting EmAGO2 protein 2.1 Experimental Methods 2.1.1 Construction of the three-dimensional structure model of the EmAGO2 protein The amino acid sequence of the EmAGO2 protein was retrieved and downloaded from the UniProt database, formatted into standard FASTA format, and imported into the AlphaFold official workflow. Combining homology sequence retrieval and template modeling results, a three-dimensional structural model of the EmAGO2 protein was generated. Five independent random seeds were used to construct the model, and the optimal conformation was selected based on pLDDT confidence. Energy minimization was performed using OpenMM, and stereochemical rationality was verified using MolProbity. Finally, high-confidence EmAGO2 three-dimensional structural coordinates were output.

[0039] 2.1.2 Compound Library Preprocessing 2D-format compounds from the Bioactive Compound Library Plus (HY-L001P) and the Fragment Compound Library (HY-L032) are preprocessed using the LigPrep module of the Schrödinger software. This preprocessing includes hydrogenation, energy optimization, format conversion, desalting, and charge correction, ultimately outputting the 3D structures of the compounds.

[0040] 2.1.3 Virtual Screening and Molecular Docking Virtual screening was conducted using the Virtual Screening Workflow module of Schrödinger software. Pre-treated compounds were imported, and molecular docking experiments were performed using the Glide module. Precise docking was achieved through geometric and energy matching between the EmAGO2 protein and the compounds. After docking, key indicators such as the binding strength between the target and the compound, and the structural stability of the compounds were manually verified to screen candidate compounds with strong binding affinity to the EmAGO2 protein.

[0041] 2.2 Experimental Results Based on the three-dimensional structure of EmAGO2 predicted by AlphaFold, a small molecule compound library was subjected to high-throughput virtual screening, followed by precise molecular docking using the Glide module. The final screening yielded Thonningianin A (Catalog_NO: HY-N4084; Docking Score: -10.759), a small molecule compound that can form a significantly high-affinity binding with EmAGO2. Its structural formula is shown below: .

[0042] 3. Experimental verification of the binding affinity between Thonningianin A and EmAGO2 protein 3.1 Micro-thermophoresis (MST) technique for detecting binding affinity 3.1.1 Experimental Methods Take 10 μL of each 50 nM EmAGO2 protein sample and mix thoroughly with equal-volume serially diluted Thonningianin A solution (starting at 5 μM, with 10 concentration gradients). Incubate at 4°C for 30 min to allow the protein and compound to fully bind and reach equilibrium. Add all incubated samples sequentially to MST-specific capillary tubes and detect using a Monolith NT.115 instrument. Detection parameters are set as follows: temperature 25°C, excitation power 40%, automatic fluorescence intensity gain adjustment, and a standard capillary tube.

[0043] 3.1.2 Experimental Results like Figure 2 As shown, microthermophoresis (MST) was used to preliminarily verify that the binding affinity constant (KD) between Thonningianin A and EmAGO2 protein was 101.3 nM, confirming that the two have a strong binding ability.

[0044] 3.2 Surface Plasmon Resonance (SPR) Technique to Verify Specific Binding 3.2.1 Experimental Methods Thonningianin A compound was diluted to a preset printing concentration of 10 μM and printed onto the surface of a 3D photocrosslinked chip using a Biodot™ AD1520 chip array printer. Four replicates were set for each sample to ensure experimental reliability. Four positive control points (rapamycin) were printed at the four corners of the chip as experimental references. After printing, the chip was dried in a vacuum environment and then placed in the photocrosslinking instrument for photocrosslinking reaction. After the reaction, the chip was washed sequentially with DMF (N,N-dimethylformamide), anhydrous ethanol (C2H5OH), and ultrapure water (H2O) for 15 minutes each to remove unbound impurities and residual reagents. After drying with nitrogen, the chip was assembled with a Flowcell Cover, sealed, and stored for subsequent interaction detection experiments.

[0045] EmAGO2 protein samples were diluted to five concentration gradients: 10 nM, 40 nM, 160 nM, 640 nM, and 2560 nM. PBST (pH 7.4, containing 0.1% Tween 20) was used as the flow carrier throughout the experiment, with all samples being flowed sequentially for testing. Analytes were administered in 0.5 μL increments. s - ¹ A flow rate of 2 μL was applied to the chip surface; in the surface regeneration stage, Glycine-HCl solution (pH=2.0) was used as the regeneration solution, and the flow rate was set to 2 μL. s - ¹. The experiment involved loading compound samples of different concentration gradients in order of increasing concentration, with the flow rate maintained at 0.5 μL. s - ¹, the reaction temperature was controlled at 4℃, the binding time was 600s, and the dissociation time was 360s.

[0046] 3.2.2 Experimental Results like Figure 3 As shown, further verification using surface plasmon resonance (SPR) technology confirmed that Thonningianin A can specifically and strongly bind to the EmAGO2 protein, with an equilibrium dissociation constant (KD) of 5.51 nM. Figure 3 A represents the binding curves of the DMSO solvent control with different concentrations of EmAGO2 protein. Figure 3 B shows the specific binding curves of Thonningianin A with different concentrations of EmAGO2 protein. The solvent control group showed no obvious binding response, further verifying the specificity of the binding between the two.

[0047] 4. In vitro inhibitory activity of Thonningianin A against Echinococcus multilocularis protocercariae 4.1 Experimental Methods Protocercariae that have been isolated and purified with an activity ≥95% were adjusted to a concentration of 1.2 × 10⁻⁶ using high-glucose DMEM medium containing 10% fetal bovine serum. 4 The concentration of Thonningianin A was measured at 0 μL / mL, followed by the addition of 100 μL (containing 120 cells) to each well of a 96-well plate. Six concentration gradients of Thonningianin A (0, 1, 2, 5, 10, and 20 μM) were established, with three replicates per group. An equal volume of DMSO was added to the solvent control group to maintain consistent concentrations across groups. After incubation at 37°C and 5% CO2 for 48 hours, the 96-well plates were observed using PI staining combined with fluorescence microscopy to count and calculate the protocercariae survival rate. Finally, the dose-response curve was fitted using GraphPad Prism software, and the half-maximal inhibitory concentration (IC50) was calculated. 50 This was used to evaluate the inhibitory activity of Thonningianin A.

[0048] 4.2 Experimental Results like Figure 4 As shown, the survival rate of protocercariae decreased significantly in a dose-dependent manner with increasing Thonningianin A concentration (0-20 μM), indicating that this compound possesses potent anti-protocercariae activity in vitro. The half-maximal inhibitory concentration (IC50) of Thonningianin A against the protocercariae of *Echinococcus multilocularis* was calculated. 50 The value was 9.22 μM.

[0049] 5. In vitro experiment on the inhibition of growth and development of multilocular echinococcosis vesicles by Thonningianin A 5.1 Experimental Methods Two hundred and ten thousand *Echinococcus multilocularis* larvae with a viability ≥95% were inoculated into cell culture flasks, and 10 mL of high-glucose DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) was added. The flasks were then incubated at 37°C in a 5% CO2 incubator. The medium was replaced with fresh medium every 7 days for 3 months until the vesicle diameter reached approximately 5 mm. The cultured vesicles were collected, counted, and seeded into groups: 50 vesicles were seeded into each well of a 6-well plate, and Thonningianin A small molecule inhibitors at final concentrations of 0 μM, 5 μM, 10 μM, and 20 μM were added, respectively. After 48 h of treatment, the morphological changes of the vesicles in each group were observed and recorded using an inverted microscope.

[0050] 5.2 Experimental Results like Figure 5 As shown, after treatment with Thonningianin A, the protocercariae of *Echinococcus multilocularis* exhibited a distinct shrinkage phenotype, and the degree of shrinkage significantly increased with increasing inhibitor concentration. These results indicate that this small-molecule inhibitor can significantly inhibit the growth and development of *Echinococcus multilocularis* protocercariae, and the inhibitory effect is clearly dose-dependent.

[0051] 6. In vivo pharmacodynamic experiment of Thonningianin A against multilocular echinococcosis. 6.1 Establishment of a mouse model of echinococcosis Using C57BL / 6J mice as the animal model, an infection model was established by intravenous injection of protocercariae via the portal vein. Gerbils infected with *Echinococcus multilocularis* were euthanized by cervical dislocation and disinfected by immersion in 75% alcohol. Under aseptic conditions, the cysts of the worms were detached, residual tissue was removed, and the cysts were repeatedly washed three times in pre-cooled PBS containing penicillin (100 U / mL) and streptomycin (100 U / mL). The cysts were then cut into small pieces, passed through an 80-mesh copper mesh, and the supernatant was discarded after the worms settled naturally. The washing process was repeated until the supernatant was clear, resulting in a protocercariae suspension of 5000 worms / mL. Twelve 8-week-old C57BL / 6 mice were selected, and each mouse was injected via the portal vein with 100 μL of the suspension (containing 500 protocercariae) to complete the infection model construction.

[0052] 6.2 Experimental Grouping and Drug Treatment 6.2.1 Experimental Grouping: After successful modeling, mice infected with *Echinococcus multilocularis* were randomly divided into three groups: the Thonningianin A small molecule inhibitor intervention group, the solvent control group, and the albendazole positive drug treatment group. 6.2.2 Drug Preparation: Thonningianin A was dissolved in DMSO to prepare a 20 mg / mL stock solution, which was then diluted with physiological saline to a working concentration of 1 mg / mL before use. 6.2.3 Administration Method and Cycle: Oral administration was performed via gavage. All drugs were dissolved in physiological saline solution containing 1% DMSO (volume fraction). The dosage was set as follows: albendazole group 40 mg / kg, Thonningianin A group 40 mg / kg. The solvent control group mice were administered an equal volume of physiological saline solution containing 1% DMSO via gavage. The gavage volume was 10 mL / kg body weight. Administered once every 3 days for 30 consecutive days (10 times in total).

[0053] 6.3 Experimental Results After treatment, the mice were euthanized and dissected. The body weight, liver weight, and weight of the removed cysts were recorded. The results are as follows: (1) In vivo safety evaluation, such as Figure 6 As shown, the body weight of mice in each group showed a slow growth trend during the treatment period, with no significant difference between groups, indicating that Thonningianin A has good in vivo safety at a dose of 40 mg / kg and no obvious systemic toxicity.

[0054] (2) Evaluation of in vivo efficacy, such as Figure 7 As shown, the diagram includes gross morphology images of the livers of mice in each group, statistical charts of liver weight, and statistical charts of echinococcosis cyst weight, which can visually demonstrate the inhibitory effect of the drug on the lesions. Compared with the solvent control group, mice treated with the small molecule inhibitor Thonningianin A at 40 mg / kg showed a significant reduction in the area of ​​lesions and a significant decrease in the number and weight of echinococcosis cysts; the therapeutic effect of 40 mg / kg Thonningianin A was comparable to that of the first-line clinical drug albendazole (40 mg / kg), confirming that Thonningianin A has good anti-hydatid echinococcosis activity in vivo.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The application of Thonningianin A in the preparation of drugs for treating echinococcosis, characterized in that, The structural formula of Thonningianin A is shown below: 。 2. The application according to claim 1, characterized in that, The Thonningianin A targets the EmAGO2 protein and binds to it with high affinity.

3. The application according to claim 2, characterized in that, The binding affinity constant KD of Thonningianin A to EmAGO2 protein is 101.3 nM, and the equilibrium dissociation constant KD is 5.51 nM.

4. The application according to claim 1, characterized in that, The aforementioned echinococcosis is caused by infection with the vesicular larvae of Echinococcus multilocularis. Its typical liver lesions are irregularly shaped white vesicle clusters or masses formed by the aggregation of a large number of small vesicles, which are poorly demarcated from the host liver tissue and exhibit infiltrative and exophytic growth.

5. The application according to claim 1, characterized in that, The drug is in the form of an oral dosage form.

6. The application according to claim 1, characterized in that, The Thonningianin A is dissolved in DMSO and diluted with physiological saline before administration, and the dosage of Thonningianin A is not less than 40 mg / kg.

7. The application according to claim 1, characterized in that, When administered orally at a dose of 40 mg / kg, Thonningianin A reduced the lesion area and significantly decreased the number and weight of echinococcosis cysts in individuals infected with multilocular echinococcosis, with therapeutic effects comparable to those of albendazole at 40 mg / kg.

8. A pharmaceutical composition for treating echinococcosis, characterized in that, It includes an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient is Thonningianin A or a pharmaceutically acceptable salt thereof or a hydrate of said salt.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition is in the form of an oral dosage form.

10. The pharmaceutical composition according to claim 9, characterized in that, The oral dosage form is selected from one of the following: tablets, granules, powders, capsules, liposome-loaded drugs, oral liquids, or lozenges.

Images