Application of all-transretinoic acid in preparation of medicine for treating echinococcosis infection

Abstract

The invention provides application of all-transretinoic acid in preparation of drugs for treating echinococcosis infection, and belongs to the technical field of biological drugs. The invention relates to an application of all-transretinoic acid in preparation of a medicine for treating echinococcosis infection or an application in preparation of a medicine for improving intestinal flora imbalance and / or immunosuppression microenvironment caused by echinococcosis infection. The all-transretinoic acid can obviously reduce the proportion of immunosuppressive cells MDSCs in the spleen of the host infected by the echinococcosis and reduce the abundance of lactobacillus in the intestinal flora of the host, so that the cyst weight is reduced, and the severity of the disease is improved. Therefore, development of drugs for targeting intestinal flora and host immunoregulation based on all-transretinoic acid provides technical support for efficient prevention and treatment of echinococcosis and other immune-related diseases, and social benefits are obvious.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to the application of all-trans retinoic acid in the preparation of drugs for Echinococcus granulosus infection. Background Technology

[0002] Echinococcosis is a disease caused by the ingestion of Echinococcus tapeworm eggs. The larvae (echinococcosis larvae) develop into cysts in organs such as the liver and lungs. Echinococcus tapeworm eggs are primarily transmitted orally; in rare cases, they can enter through inhalation (e.g., from dust containing eggs) or skin wounds. Individuals with frequent contact with canines are susceptible. Commonly used treatments for Echinococcosis include the broad-spectrum anthelmintics albendazole and mebendazole.

[0003] All-trans retinoic acid (ATA) is an active metabolite of vitamin A. It exerts its pharmacological effects by regulating cell differentiation, proliferation, and apoptosis. Clinically, it is mainly used to treat hematological diseases, such as acute promyelocytic leukemia (APL), and can also be used as adjuvant therapy for some skin diseases and malignant tumors. Currently, there are no reports on the use of ATA for parasitic diseases. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a new use of all-trans retinoic acid, namely, the application of all-trans retinoic acid in the preparation of drugs for Echinococcus larvae infection.

[0005] This invention provides the application of all-trans retinoic acid in the preparation of drugs for Echinococcus granulosus infection.

[0006] Preferably, the echinococcosis larvae in the echinococcosis infection include multilocular echinococcosis larvae and / or fine echinococcosis larvae.

[0007] This invention provides the application of all-trans retinoic acid in the preparation of drugs that improve intestinal flora imbalance caused by Echinococcus granulosus infection.

[0008] This invention provides the application of all-trans retinoic acid in the preparation of drugs that improve the immunosuppressive microenvironment caused by Echinococcus granulosus infection.

[0009] Preferably, the drug comprises at least one of the following dosage forms: oral, injectable, spray, and suppository.

[0010] Preferably, the oral preparation includes at least one of the following: tablets, capsules, drops, and powders.

[0011] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0012] Preferably, the pharmaceutically acceptable excipients include at least one of the following: fillers, diluents, binders, wetting agents, flavoring agents, antioxidants, disintegrants, lubricants, flow aids, effervescent agents, colorants, dispersants, film-forming agents, plasticizers, pore-forming agents, light-blocking agents, pH adjusters, osmotic pressure adjusters, integrators, and thickeners.

[0013] Preferably, the mass percentage of all-trans retinoic acid in the drug is ≥98.0%.

[0014] This invention provides the application of all-trans retinoic acid in the preparation of drugs for Echinococcus granulosus infection. Using mice infected with Echinococcus granulosus as the research subject, this invention analyzed the therapeutic efficacy of all-trans retinoic acid against Echinococcus granulosus infection. Experimental results showed that all-trans retinoic acid effectively inhibited the size and weight of multilocular Echinococcus granulosus cysts, significantly reduced the proportion of bone marrow-derived suppressor cells in the spleen of infected mice, and significantly reduced the abundance of lactobacilli positively correlated with Echinococcus granulosus infection in the mouse intestine. Therefore, the application scheme provided by this invention will support the development of drugs targeting the intestinal flora and host immune regulation, promote the research and development of novel strategies for the prevention and control of parasitic diseases such as echinococcosis, and provide a theoretical basis for protecting the life, health, and property safety of herders, resulting in significant social benefits. Attached Figure Description

[0015] Figure 1 The results show the regulation of the proportion of bone marrow-derived suppressor cells in the spleen of mice infected with Echinococcus multilocularis. Figure 2 The results show the effects of all-trans retinoic acid on the gut microbiota of mice infected with Echinococcus multilocularis. Figure 3 The effects of different concentrations of all-trans retinoic acid (intraperitoneal injection) on the size and weight of cysts in mice infected with Echinococcus granulosus are shown in Figure a. The experimental procedure is shown in Figure b. The size and morphology of cysts in different treatment groups are shown in Figure c. The statistical results of cyst weight in different treatment groups are shown in Figure c. Figure 4 The results show the efficacy of dietary intervention containing all-trans retinoic acid on mice infected with Echinococcus multilocularis. Detailed Implementation

[0016] This invention provides the application of all-trans retinoic acid in the preparation of drugs for Echinococcus granulosus infection.

[0017] In this invention, the all-trans retinoic acid includes C 20 H 28 O2, CAS: 302-79-4, structural formula as shown in Formula I. The all-trans retinoic acid described in this embodiment of the invention was purchased from Solarbio, catalog number A9120.

[0018] Formula I.

[0019] In this invention, the echinococcosis larvae in the echinococcosis infection preferably include *Echinococcus multilocularis* and / or *Echinococcus granulosus*. Both *Echinococcus multilocularis* and *Echinococcus granulosus* are common pathogenic parasites causing echinococcosis infection, and both can colonize the liver or lungs. Rupture of the formed cysts can trigger anaphylactic shock and immune infection, and can also affect intestinal flora imbalance and create an immunosuppressive microenvironment.

[0020] In this invention, the drug can effectively inhibit the colonization of cysts caused by Echinococcus larvae infection through injection or dietary intervention, thereby reducing the volume and quality of cysts in the body.

[0021] This invention provides the application of all-trans retinoic acid in the preparation of drugs that improve intestinal flora imbalance caused by Echinococcus granulosus infection.

[0022] In this invention, the intestinal flora imbalance caused by Echinococcus infection preferably includes a significant upregulation of Lactobacillus. The drug, administered intraperitoneally or through dietary intervention, can significantly reduce the abundance of Lactobacillus, thereby exerting a targeted regulatory effect on the host's intestinal flora.

[0023] This invention provides the application of all-trans retinoic acid in the preparation of drugs that improve the immunosuppressive microenvironment caused by Echinococcus granulosus infection.

[0024] In this invention, the improvement of the immunosuppressive microenvironment caused by Echinococcus granulosus infection preferably includes all-trans retinoic acid (ATA) through dietary intervention, which can effectively reverse the increase in the content of bone marrow-derived suppressor cells (MDSCs) in the spleen of infected hosts. In one embodiment of this invention, compared with a basal diet, the ATRA synthetic diet significantly reduced the proportion of MDSCs in the spleen of mice, indicating that ATA can improve the Echinococcus granulosus infection status in mice by modulating host immunity.

[0025] In this invention, the drug preferably comprises at least one of the following dosage forms: oral dosage form, injection, spray, and suppository. The oral dosage form preferably comprises at least one of the following: tablet, capsule, drops, and powder. The injection preferably comprises injectable powder, injectable solution, or drops.

[0026] In the present invention, the drug preferably further comprises pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients preferably include at least one of the following: fillers, diluents, binders, wetting agents, flavoring agents, antioxidants, disintegrants, lubricants, glidants, effervescent agents, coloring agents, dispersants, film-forming agents, plasticizers, pore-forming agents, light-shielding agents, pH regulators, osmotic pressure regulators, chelating agents, and thickening agents. The drug is conventionally prepared with appropriate excipients according to the type of dosage form. There is no particular limitation on the preparation method of the drug in the present invention, and drugs well-known in the art can be used. The mass percentage of all-trans retinoic acid in the drug is preferably 5% - 95%, and can be 8% - 90%, can also be 10% - 85%, can further be 15% - 80%, further can be 20% - 75%, more can be 30% - 60%, and further can be 40% - 50%.

[0027] In the present invention, the drug can be used against echinococcus infections occurring in animals or humans. The animals preferably include canines, bovines, sheep, pigs, etc.

[0028] The following examples are used to illustrate in detail the application of all-trans retinoic acid provided by the present invention in the preparation of drugs for echinococcus infections, but they should not be construed as limiting the protection scope of the present invention.

[0029] Example 1 Effect of all-trans retinoic acid (ATRA) on mouse bone marrow-derived suppressor cells 1. Establishment and grouping of animal models Animals: Healthy SPF-grade C57BL / 6J female mice (Vital River, license SCXK Jing 2021-0006), weighing 20 ± 2 g.<*

[0030] Grouping: Divided into an uninfected group (Naive), a normal diet group (Normal diet), and an ATRA diet group (ATRAdiet), with at least 6 mice in each group.

[0031] Feed: Normal diet: Sterile mouse and rat maintenance feed (Keao Xieli, batch number 25043221). Synthetic feed: Sterile irradiated feed (Keao Xieli, batch number N2025041404), with the ATRA content (100 mg / kg) verified by HPLC (error < 5%).

[0032] 2. Specific steps (1) The uninfected group and the normal diet group were fed normal feed, and the ATRA diet group was fed ATRA synthetic feed for a total of 4 weeks.

[0033] (2) Except for the uninfected group, each group of mice was intraperitoneally injected with <2000> protoscoleces, and the control group was injected with an equal amount of PBS.

[0034] (3) Infection lasted for 3 months.

[0035] 3. Detection index: Analysis of the content of spleen bone marrow-derived suppressor cells (MDSCs) Mouse spleens were collected, washed once with PBS, and placed on a 70 μm cell strainer. Using a 5 mL sterile syringe with a rubber stopper, the spleen tissue was repeatedly ground into fragments. The fragments were then rinsed with PBS into a 50 mL collection tube, collecting a total of 10 mL of cell suspension. The spleen cells were centrifuged to a concentration of 2 × 10⁻⁶ cells / mL. 6 / EP tubes were used to detect CD45 antibodies, CD11b antibodies, and GR-1 antibodies using flow cytometry. + CD11b + GR-1 + Cell ratio, i.e., bone marrow-derived suppressor cells.

[0036] The results are as follows Figure 1 As shown, compared with the uninfected group, the proportion of MDSCs in infected mice was significantly increased. At the same time, compared with the basal diet, the ATRA synthetic diet significantly reduced the proportion of MDSCs in the spleen of mice, indicating that it can improve the infection status of Echinococcus larvae in mice by regulating host immunity.

[0037] Example 2 Effects of all-trans retinoic acid on gut microbiota in mice infected with Echinococcus multilocularis Two groups of mice were set up, with 6 mice in each group: the ATRA diet group was fed ATRA synthetic feed for 4 weeks; the normal diet control group was fed normal feed for 4 weeks; after 4 weeks, 2000 protostomes were injected intraperitoneally into both groups of mice.

[0038] Fresh feces were collected from each group of mice. Fecal DNA was extracted using a DNA extraction kit (TIANGEN, DP328). The concentration was determined using Nanodrop 2000 and serially diluted. qPCR was then performed using Lactobacillus-specific primers (LabF362: 5'-AGCAGTAGGGAATCTTCCA-3', SEQ ID NO:1; LabR677: 5'-CACCGCTACACATGGAG-3', SEQ ID NO:2). The reaction program used fecal DNA as a template and employed a two-step qPCR method for detection. The system is shown in Table 1.

[0039] Table 1 Reaction System

[0040] The amplification program included: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 15 s, annealing at 60℃ for 1 min, for 40 cycles. Melting curves were set at 95℃ for 15 s, 60℃ for 1 min, and 95℃ for 15 s. Standard curves were plotted based on the DNA concentration after different bacterial dilutions and their corresponding Ct values ​​(threshold cycle number). Based on these curves, the abundance of *Lactobacillus* in the mouse gut in different groups was calculated.

[0041] See results Figure 2 Compared with the control group, intraperitoneal administration of all-trans retinoic acid and / or dietary intervention significantly reduced the abundance of lactobacilli in the feces of infected mice, indicating that the drug also has a regulatory effect on the host gut microbiota.

[0042] Example 3 The effect of all-trans retinoic acid administered intraperitoneally on Echinococcus multilocularis cysts in mice Gerbils infected with *Echinococcus multilocularis* were euthanized by cervical dislocation. The gerbils were then sterilized by spraying their surface thoroughly with 75% alcohol and transferred to a laminar flow hood for aseptic isolation of the protoscolex. *Echinococcus multilocularis* cysts were detached from the gerbil tissue, and any remaining tissue was removed. The cysts were placed in pre-cooled PBS buffer (containing 100 U / mL penicillin and 100 U / mL streptomycin) and washed three times. The cyst tissue was minced with 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 were observed to be plump, oval-shaped, clearly structured, and exhibiting obvious peristalsis. Protoscolex were intraperitoneally infected into 7-week-old C57 / BL6J mice at a rate of 2000 worms / mouse. Treatment experiments were conducted 30 days after infection.

[0043] Experimental groups: solvent control group (DMSO:corn oil = 1:9, volume ratio), 12.5 mg / kg ATRA group, 25 mg / kg ATRA group, and 50 mg / kg ATRA group. Mice in each group were intraperitoneally injected with 100 μL of the drug or solvent control every 3 days for 30 consecutive days. The procedure is as follows: Figure 3 a. After treatment, mice were euthanized by cervical dislocation, and the size and / or weight of the multilocular echinococcosis cysts were measured to analyze the therapeutic effects of different drug doses on echinococcosis infection in mice.

[0044] Data analysis methods: After isolating Echinococcus multilocularis cysts from mice, the size of the cysts in each group of mice was initially determined by visual inspection. The weight of the cysts of all mice was measured using a precision analytical balance, and a one-way ANOVA was performed.

[0045] See results Figure 3In samples b and c, compared with the solvent control group, the weight of Echinococcus polymorphon cysts in mice decreased most significantly at a drug concentration of 12.5 mg / kg.

[0046] Example 4 The efficacy of all-trans retinoic acid dietary intervention on mice infected with Echinococcus multilocularis. Experimental grouping: Same as in Example 1, divided into normal diet group and ATRA diet group (ATRA content in feed is 100 mg / kg), each mouse was fed about 5 g of feed per day.

[0047] Experimental procedure: After being fed different diets for 4 weeks, each mouse was intraperitoneally injected with 2000 protoscolex segments and infected for 3 months, during which time the same diet was maintained. 3 months after infection, the mice were euthanized by cervical dislocation, and the cysts attached to the intestines and other organs were carefully removed from the abdominal cavity and weighed (g).

[0048] Statistical methods: Data were plotted using Prism 8 software (Graphpad Prism 8.4.3, La Jolla, CA). Data are presented as mean ± standard deviation (Mean ± SD). Differences between groups were analyzed using a two-tailed t-test, and p < 0.05 was considered statistically significant.

[0049] See results Figure 4 Compared with the normal diet group, the number of cysts in mice in the synthetic diet group containing 100 mg / kg ATRA was significantly reduced, indicating that all-trans retinoic acid can reduce the number of Echinococcus multilocularis larvae in mice.

[0050] The results of the above embodiments show that all-trans retinoic acid can significantly downregulate the proportion of immunosuppressive microcellular carcinoma cells (MDSCs) in the spleen of mice infected with Echinococcosis and reduce the abundance of lactobacilli in the host gut microbiota, thereby reducing cyst weight and improving disease severity. These results provide experimental evidence for the development of drugs based on all-trans retinoic acid that target gut microbiota and host immune regulation, and provide technical support for the efficient prevention and treatment of immune-related diseases such as echinococcosis, with significant social benefits.

[0051] 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. Application of all-trans retinoic acid in the preparation of drugs for Echinococcus larvae infection.

2. The application according to claim 1, characterized in that, The echinococcosis infection mentioned above includes echinococcosis multilocularis and / or echinococcosis granulosus.

3. Application of all-trans retinoic acid in the preparation of drugs to improve intestinal flora imbalance caused by Echinococcus larvae infection.

4. Application of all-trans retinoic acid in the preparation of drugs that improve the immunosuppressive microenvironment caused by Echinococcus granulosus infection.

5. The application according to any one of claims 1 to 4, characterized in that, The drug includes at least one of the following dosage forms: oral, injectable, spray, and suppository.

6. The application according to claim 5, characterized in that, The oral dosage form includes at least one of the following: tablets, capsules, drops, and powders.

7. The application according to any one of claims 1 to 4, characterized in that, The drug also includes pharmaceutically acceptable excipients.

8. The application according to claim 7, characterized in that, The pharmaceutically acceptable excipients include at least one of the following: fillers, diluents, binders, wetting agents, flavoring agents, antioxidants, disintegrants, lubricants, flow aids, effervescent agents, colorants, dispersants, film-forming agents, plasticizers, pore-forming agents, opacifiers, pH adjusters, osmotic pressure adjusters, integrators, and thickeners.

9. The application according to claim 7, characterized in that, The mass percentage of all-trans retinoic acid in the drug is 5% to 95%.

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