Use of toll-like receptor agonist resiquimod in the preparation of a drug for treating echinococcosis

Abstract

The application relates to the technical field of echinococcosis medicines, and is an application of a Toll-like receptor agonist Resiquimod in preparation of an echinococcosis treatment medicine. The application discloses, for the first time, the application of the Toll-like receptor agonist Resiquimod in preparation of the echinococcosis treatment medicine. The Toll-like receptor agonist Resiquimod can promote the fibrosis of the tissue around a lesion of a mouse infected with multilocular metacestode and further inhibit the growth of the multilocular metacestode, and reduce the lesion volume of the liver of the mouse infected with the multilocular metacestode, thereby providing a new way for echinococcosis treatment.

Description

Technical Field

[0001] This invention relates to the field of echinococcosis drug technology, specifically the application of the Toll-like receptor agonist Resiquimod in the preparation of drugs for treating echinococcosis. Background Technology

[0002] Echinococcosis is a serious public health problem prevalent worldwide. Alveolar echinococcosis (AE), in particular, is caused by the parasitic larvae (ecchymocarcinoma larvae) of the Echinococcus multilocularis (Em) in humans, almost always originating in the liver. This disease has an insidious onset, rapid progression, and resembles a malignant tumor. It can metastasize from the liver to other vital organs via the blood and lymphatic system, hence the nickname "parasitic cancer." The 10-year mortality rate for untreated alveolar echinococcosis is as high as 94%.

[0003] The parasitic infestation of the liver by *Echinococcus multilocularis* and the formation of space-occupying lesions is a gradual process. As the disease progresses, a fibrous capsule formed by the host's fibroblasts encapsulates the echinococcosis, creating a foreign body granuloma. Persistent chronic inflammation induces liver fibrosis. On one hand, excessive fibrosis damages the liver parenchyma, eventually leading to the collapse of the parenchyma and its replacement by collagen-rich tissue, resulting in loss of liver function. On the other hand, localized liver fibrosis can further limit the invasive spread of the parasite's lesions.

[0004] Resiquimod (R-848), a Toll-like receptor agonist, is a small molecule compound of the imidazoquinone class that activates Toll-like receptors 7 and 8 (TLR7 / 8), thereby inducing immune cells to produce cytokines such as TNF-α, IL-6, and IFN-α. Current research indicates that Resiquimod (R-848) possesses antiviral and antitumor effects, inducing antitumor responses and reducing cachexia. In a mouse model of pancreatic ductal adenocarcinoma (PDAC), Resiquimod (R-848) inhibited tumor growth and metastasis through the TLR7 / 8 signaling pathway, while simultaneously improving body weight and serum albumin levels. Furthermore, studies on Schistosoma mansoni have suggested that R848 may serve as a novel therapeutic or vaccine immunostimulant for Schistosoma mansoni infection. Summary of the Invention

[0005] This invention provides the application of the Toll-like receptor agonist Resiquimod in the preparation of drugs for treating echinococcosis, overcoming the shortcomings of the prior art. It discloses for the first time the application of the Toll-like receptor agonist Resiquimod in the preparation of drugs for treating echinococcosis, which can promote fibrosis of the tissue around the lesions of Echinococcus multilocularis in mice, thereby inhibiting the growth of Echinococcus multilocularis and reducing the volume of liver lesions in mice infected with Echinococcus multilocularis.

[0006] The technical solution of the present invention is achieved through the following measures: the application of a Toll-like receptor agonist, Resiquimod, in the preparation of a drug for treating echinococcosis.

[0007] The following are further optimizations and / or improvements to the above-mentioned technical solution:

[0008] The Toll-like receptor agonist Resiquimod, as the active ingredient of the drug, has the following structural formula:

[0009] .

[0010] The echinococcosis mentioned above is alveolar echinococcosis, which is a disease caused by infection with Echinococcus multilocularis.

[0011] The aforementioned drugs also include pharmaceutically acceptable carriers or excipients.

[0012] The dosage forms of the above-mentioned drugs are pharmaceutically acceptable, including tablets, granules, capsules, and oral solutions.

[0013] The aforementioned Toll-like receptor agonist Resiquimod inhibits the expansion and growth of Echinococcus multilocularis by promoting fibrosis in the tissue surrounding the lesions of Echinococcus multilocularis infection.

[0014] The Toll-like receptor agonist Resiquimod was administered at a concentration of 2 mg / kg to inhibit echinococcosis in mice via intraperitoneal injection.

[0015] This invention discloses for the first time the application of the Toll-like receptor agonist Resiquimod in the preparation of drugs for treating echinococcosis. Resiquimod can promote fibrosis of tissues around lesions of Echinococcus multilocularis in mice, thereby inhibiting the growth of Echinococcus multilocularis and reducing the volume of liver lesions in mice infected with Echinococcus multilocularis, providing a new approach for the treatment of echinococcosis. Attached Figure Description

[0016] Figure 1 This is an HE staining image of liver tissue from mice in the model group of this invention.

[0017] Figure 2This is a comparison of the size of liver lesions in mice in the Resiquimod treatment group and the control group according to the present invention.

[0018] Figure 3 Masson staining images of liver tissue from mice in the Resiquimod treatment group and control group of this invention. Detailed Implementation

[0019] This invention is not limited to the following embodiments; specific implementation methods can be determined based on the technical solution of this invention and actual circumstances. Unless otherwise specified, all chemical reagents and chemical products mentioned in this invention are well-known and commonly used chemical reagents and chemical products in the prior art.

[0020] The present invention will be further described below with reference to embodiments:

[0021] Example 1: Application of the Toll-like receptor agonist Resiquimod in the preparation of drugs for treating echinococcosis.

[0022] In this invention, the Toll-like receptor agonist Resiquimod is Resiquimod S8133 provided by Selleck.

[0023] Example 2: As an optimization of the above examples, the Toll-like receptor agonist Resiquimod is used as the active ingredient of the drug, and its structural formula is as follows:

[0024] .

[0025] Example 3: As an optimization of the above example, echinococcosis is alveolar echinococcosis, which is a disease caused by infection with *Echinococcus multilocularis*. *Echinococcus multilocularis* refers to one or more of the following: *Echinococcus multilocularis* larva, *Echinococcus multilocularis* embryonic cells, and *Echinococcus multilocularis* germinal layers. The *Echinococcus multilocularis* larva is a *Echinococcus multilocularis* larva, also known as a vesicular larva.

[0026] Example 4: As an optimization of the above examples, the drug also includes a pharmaceutically acceptable carrier or excipient.

[0027] Example 5: As an optimization of the above examples, the dosage form of the drug is a pharmaceutically acceptable dosage form, which includes tablets, granules, capsules and oral liquids.

[0028] Example 6: As an optimization of the above examples, the Toll-like receptor agonist Resiquimod inhibits the expansion and growth of Echinococcus multilocularis by promoting fibrosis in the tissue surrounding the lesions of Echinococcus multilocularis infection.

[0029] Example 7: As an optimization of the above example, the concentration of the Toll-like receptor agonist Resiquimod used to inhibit echinococcosis in mice was 2 mg / kg, and the administration method was intraperitoneal injection.

[0030] Example 8: Investigating the effect of the Toll-like receptor agonist Resiquimod (hereinafter referred to as Resiquimod) on mice infected with vesicular larvae.

[0031] Collection of Protostomia vesicolae: Gerbils infected with vesicolae in the peritoneum for at least 6 months were selected, euthanized by cervical dislocation, and immersed in 75% ethanol for 3 to 5 minutes. The peritoneal lesion tissue of the mice was isolated, placed in a culture dish containing PBS buffer, and washed 2 to 3 times. The lesion tissue was cut and placed in a 10 mL cross-handled glass homogenizer for thorough grinding. The tissue homogenate was poured into a 100-mesh cell sieve for filtration to collect the Protostomia vesicolae. Eosin staining was used to determine the viability.

[0032] Establishment of a mouse model of *Echinococcus faecium* infection: The concentration of *Echinococcus faecium* protoscolex was adjusted to a total volume of 100 μL. Healthy 6- to 8-week-old female C57BL / 6 mice were selected. After anesthesia, the liver was exposed by dissecting along the midline of the abdomen. *Echinococcus faecium* protoscolex was aspirated with a sterile syringe and injected subcapsularly into the liver. The puncture site was pressed with a dry cotton swab until hemostasis was achieved. The peritoneal cavity was gently rinsed with sterile PBS and then sutured. The mice were placed in an incubator after surgery to observe their condition. After the mice regained consciousness, they were returned to their cages for normal rearing, thus obtaining the model group mice.

[0033] Administration: Mice in the model group were fed normally. At week 8, the mice were randomly divided into a Resiquimod treatment group and a control group. Mice in the Resiquimod treatment group were given a dose of 2 mg / kg. Resiquimod was dissolved in DMSO, diluted with physiological saline, and administered intraperitoneally. The dose was given once every 3 days for 12 weeks. At the same time, the control group was given an equal volume of DMSO physiological saline solution without Resiquimod. After the administration was completed, the mice in each group were fed normally for another 4 weeks.

[0034] Experiment 1: HE staining of mouse liver tissue infected with cysticercosis

[0035] Mice from the successfully modeled group had their livers collected. The livers were fixed in 10% neutral formalin, embedded in paraffin, and sectioned to a thickness of 4 μm. Liver tissue approximately 1 cm × 1 cm × 1 cm in size was selected from 0.5 cm around the lesion as the close liver tissue (CLT). Simultaneously, liver tissue approximately 1 cm × 1 cm × 1 cm in size was collected from approximately 2 to 3 cm away from the lesion as the distance liver tissue (DLT).

[0036] Tissue samples were collected from the area surrounding and distal to the lesion. After routine dewaxing, the sections were immersed in hematoxylin staining solution for 3 to 5 minutes, rinsed with running water, differentiated with acid-alcohol for 5 to 10 seconds, rinsed with running water, blue-inverted in distilled water for 10 minutes, stained with eosin for 2 to 3 minutes, rinsed with running water, and dehydrated with a series of alcohols. After clearing with xylene, the sections were mounted with resin for preservation. HE staining results are shown below. Figure 1 As shown, by Figure 1 It can be seen that there are a large number of inflammatory cells infiltrating around the lesions in the mouse model infected with cysticercosis, indicating that the mouse model was successfully established.

[0037] Experiment 2: Investigating the effect of Resiquimod on the size of lesions in mice infected with vesicular larvae.

[0038] Liver samples were collected from mice in the Resiquimod treatment group and the control group, and mouse body weight and liver weight were recorded. The size of the liver lesions in each group was observed. The lesion size results are as follows: Figure 2 As shown, by Figure 2 It can be seen that the lesion volume in the Resiquimod treatment group was significantly smaller than that in the control group. This indicates that Resiquimod treatment significantly reduced the parasite burden on the liver of mice infected with *Echinococcus vesicator*.

[0039] Experiment 3: Masson staining test on mouse liver tissue infected with vesicular larvae.

[0040] Mice in the Resiquimod treatment group and control group had their liver tissue collected, fixed, embedded in paraffin, sectioned, and routinely dewaxed. The tissue was then mordanted and incubated overnight at room temperature or for 1 hour at 57-60°C, followed by rinsing with running water for 10 minutes. Azurite blue was then stained for 2-3 minutes, followed by rinsing with running water for 10-15 seconds. Mayer hematoxylin was stained for 2-3 minutes, followed by rinsing with running water for 10-15 seconds. Differentiation was halted by acidic ethanol until the tissue turned red, followed by rinsing with water to terminate differentiation. Ponceau fuchsin was stained for 10 minutes, followed by rinsing with running water for 10-15 seconds. The tissue was treated with phosphomolybdic acid solution for 10 minutes, the supernatant was discarded, and aniline blue staining solution was added directly for 5 minutes. The aniline blue staining solution was rinsed with weak acid, followed by further weak acid treatment for 2 minutes. The tissue was dehydrated with a gradient of alcohols, cleared with xylene, and mounted with resin for preservation.

[0041] The results after Masson staining are as follows Figure 3 As shown, by Figure 3 As can be seen, compared with the control group, the Resiquimod treatment group showed enhanced fibrosis in the tissues surrounding the lesions. This indicates that Resiquimod inhibits the expansion and growth of Echinococcus multilocularis by promoting fibrosis in the tissues surrounding the lesions of mice infected with Echinococcus multilocularis.

[0042] In summary, this invention discloses for the first time the application of Resiquimod in the preparation of drugs for treating echinococcosis. It can promote fibrosis of tissues around lesions of Echinococcus multilocularis in mice, thereby inhibiting the growth of Echinococcus multilocularis and reducing the volume of liver lesions in mice infected with Echinococcus multilocularis, thus providing a new approach for the treatment of echinococcosis.

[0043] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Unnecessary technical features can be added or removed according to actual needs to meet the needs of different situations.

Claims

1. The application of Resiquimod, a Toll-like receptor agonist, in the preparation of drugs for treating alveolar echinococcosis.

2. The use of the Toll-like receptor agonist Resiquimod according to claim 1 in the preparation of a drug for treating alveolar echinococcosis, characterized in that... The Toll-like receptor agonist Resiquimod, as the active ingredient of the drug, has the following structural formula: 。 3. The use of the Toll-like receptor agonist Resiquimod according to claim 1 or 2 in the preparation of a drug for treating alveolar echinococcosis, characterized in that... Alveolar echinococcosis is a disease caused by infection with Echinococcus multilocularis.

4. The use of the Toll-like receptor agonist Resiquimod according to claim 1 or 2 in the preparation of a drug for treating alveolar echinococcosis, characterized in that... Drugs also include pharmaceutically acceptable carriers.

5. The use of the Toll-like receptor agonist Resiquimod according to claim 3 in the preparation of a drug for treating alveolar echinococcosis, characterized in that... Drugs also include pharmaceutically acceptable carriers.

6. The use of Resiquimod, a Toll-like receptor agonist according to claim 1, 2, or 5, in the preparation of a drug for treating alveolar echinococcosis, characterized in that... The dosage form of the drug is a pharmaceutically acceptable dosage form, which includes tablets, granules, capsules, and oral solutions.

7. The use of the Toll-like receptor agonist Resiquimod according to claim 3 in the preparation of a drug for treating alveolar echinococcosis, characterized in that... The dosage form of the drug is a pharmaceutically acceptable dosage form, which includes tablets, granules, capsules, and oral solutions.

8. The use of the Toll-like receptor agonist Resiquimod according to claim 4 in the preparation of a drug for treating alveolar echinococcosis, characterized in that... The dosage form of the drug is a pharmaceutically acceptable dosage form, which includes tablets, granules, capsules, and oral solutions.

9. The use of the Toll-like receptor agonist Resiquimod according to claim 1, 2, 5, 7, or 8 in the preparation of a drug for treating alveolar echinococcosis, characterized in that... The Toll-like receptor agonist Resiquimod inhibits the expansion and growth of Echinococcus multilocularis by promoting fibrosis in the tissue surrounding the lesions of Echinococcus multilocularis infection in alveolar echinococcosis.

10. The use of the Toll-like receptor agonist Resiquimod according to claim 9 in the preparation of a drug for treating alveolar echinococcosis, characterized in that... The Toll-like receptor agonist Resiquimod was administered to mice infected with alveolar echinococcosis at a concentration of 2 mg / kg via intraperitoneal injection.

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