Application of compound in resisting nematode infection and inhibiting nematode egg incubation
By screening compounds targeting the FAR-1 protein of Brazilian yennematode, the compound 2-(3-Chlorophenyl)-3-[(4-methylcyclohexyl)amino]-1H-pyrazolo[1,5-A]imidazole-7-carbonitrile was developed, which solved the drug resistance and larval removal problems of existing anti-nematode drugs, and achieved safe and efficient nematode infection prevention and control effects.
Patent Information
- Application Number
- CN202510721844.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-30
- Publication Date
- 2025-08-12
AI Technical Summary
Existing antinematode drugs have defects such as drug resistance, host immune dependence and difficulty in removing the larval stage, and it is necessary to develop safer and more efficient compounds to prevent and control nematode infection.
By screening compounds targeting the FAR-1 protein of Brazilian yennematode, the compound 2-(3-Chlorophenyl)-3-[(4-methylcyclohexyl)amino]-1H-pyrazolo[1,5-A]imidazole-7-carbonitrile was developed, which was used to inhibit nematode growth, development, and egg hatching, and reduce nematode number and lesions.
The compound significantly inhibits the hatching of nematode eggs in Brazilian yen, reduces the amount of intestinal worms, improves intestinal lesions, has good biosafety, does not affect mouse growth, and provides more effective drugs for anti-nematode infection.
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Figure CN120459088A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of biomedicine technology, and in particular to the use of compounds in resisting nematode infection and inhibiting nematode egg hatching. Background Art
[0002] Nematode disease, caused by soil-transmitted nematodes, is one of the most common diseases in the world and has the highest number of cases among neglected tropical diseases. Nematodes can infect humans, wild animals, and domestic animals, causing anemia, malnutrition, and malabsorption in the host, affecting the host's growth and development, and even leading to death. Nippostrongylus brasiliensis ) is a soil-borne intestinal parasitic nematode that primarily infects rodents (such as rats and mice). Because it shares a similar life cycle to hookworms, it is also known as rat hookworm, making it a good model for studying hookworms. The eggs of the Brazilian roundworm in the host's feces undergo two molts and develop into infective L3 larvae. Subsequently, the L3 larvae percutaneously infect mice to enter the parasitic life stage in the body, growing and developing through the respiratory and digestive tracts, and finally developing into dioecious adults in the small intestine and starting to excrete eggs on the sixth day after infection. It mainly parasitizes the host's small intestine, causing intestinal inflammation, bleeding, villous atrophy and protein loss, leading to anemia, weight loss and immune response activation in the host.
[0003] Benzimidazoles, such as albendazole and mebendazole, are commonly used to control soil-transmitted nematode infections. These drugs primarily inhibit tubulin polymerization, disrupting worm energy metabolism. However, they require multiple doses, have limited efficacy with a single dose, and can develop drug resistance. Macrolides, such as ivermectin, paralyze the worms by activating glutamate-gated chloride channels. However, these drugs are more effective against adult worms, but have limited effects on larvae and eggs, and may pose a neurotoxic risk in immunocompromised hosts. Existing drugs suffer from limitations such as resistance (long-term use of benzimidazoles can lead to drug resistance in worm strains), host immune dependency (drug efficacy may depend on the host's immune status, such as reduced infection clearance in immunocompromised animals), and difficulty clearing the larval stage (most drugs are ineffective against migratory larvae). Therefore, the development of new compounds that can safely and effectively control nematode infections is crucial and crucial for the development of anti-nematode drugs. Summary of the Invention
[0004] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of existing anti-nematode drugs and provide the application of compounds in resisting nematode infection and inhibiting nematode egg hatching.
[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions: The present invention provides the use of a compound in the preparation of a drug for resisting nematode infection. The compound is named 2-(3-Chlorophenyl)-3-[(4-methylcyclohexyl)amino]-1H-pyrazolo[1,5-A]imidazole-7-carbonitrile in English, CAS No.: 891506-09-5, and has the structural formula:
[0006] FAR-1 is a key gene in nematode development and a nematode-specific protein, making it a potential target for anti-nematode drugs. Screening for compounds that bind to FAR-1 has the potential to develop novel, low-toxic anti-nematode drugs. Building on previous screening studies, this study identified compounds that bind to the FAR-1 protein of N. brasiliensis. These compounds were shown to inhibit nematode growth and development, demonstrating strong resistance to N. brasiliensis infection. In vitro studies demonstrated that the compounds inhibited the hatching of N. brasiliensis eggs, thereby inhibiting the reproduction of the nematode. Furthermore, the compounds exhibited minimal inhibition of HEK-293T cell proliferation, demonstrating good cytotoxicity. In vivo studies demonstrated that the compounds inhibited N. brasiliensis growth in mice, reduced the number of nematodes in the host intestine, and decreased the number of eggs in the host feces. Furthermore, the compounds improved intestinal lesions in infected mice, demonstrating a moderate resistance to N. brasiliensis infection without affecting normal growth, demonstrating good biosafety. Therefore, the present invention provides a new application of the compound in resisting infection by the Brazilian nematode, provides new ideas and methods for the preparation and development of more anti-nematode drugs, and has good application prospects in the research and development of drugs for the treatment or prevention of nematode infections.
[0007] The present invention provides use of a compound in preparing a drug for resisting nematode infection.
[0008] The present invention provides use of a compound in inhibiting the growth of nematodes.
[0009] The present invention provides use of a compound in preparing a product for inhibiting the growth and development of nematodes.
[0010] The present invention provides use of a compound in inhibiting the hatching of nematode eggs.
[0011] The present invention provides use of a compound in preparing a product for inhibiting the hatching of nematode eggs.
[0012] The present invention provides use of a compound in preparing a medicine for alleviating pathological changes in a nematode parasitic site.
[0013] Preferably, the nematode is N. brasiliensis.
[0014] Furthermore, the drug can reduce the number of nematodes.
[0015] Furthermore, the drug can reduce the number of nematode eggs.
[0016] Preferably, the drug further comprises a pharmaceutically acceptable carrier or excipient.
[0017] Preferably, the dosage form of the drug is tablet, capsule, oral liquid preparation, spray or injection.
[0018] The present invention has the following beneficial effects: The present invention provides the use of a compound in resisting infection by the Brazilian nematode and inhibiting the hatching of worm eggs. Studies have shown that the compound can significantly inhibit the hatching of Brazilian nematode eggs. At the same time, the compound can inhibit the growth and development of Brazilian nematodes in mice, has a significant anti-nematode infection effect, and can resist Brazilian nematode infection. In addition, the compound has good biosafety, is non-cytotoxic, and does not affect the growth of mice, providing more effective drugs for resisting nematode infections, and can be used to prepare and develop more nematode growth inhibition products and anti-nematode infection drugs, providing methods and products for developing more anti-nematode infection drugs. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 The graph shows the effect of the compound on the hatching of Nippostrongylus brasiliensis eggs (the compound numbered E002-0872 in the figure is the same below).
[0020] Figure 2 Figure 2 shows the number of N. brasiliensis in mice treated with the compound.
[0021] Figure 3 Figure 2 is a graph showing the number of worm eggs in the feces of mice treated with the compound.
[0022] Figure 4 Figure 2 is the ratio of intestinal villus length to crypt depth in mice infected with N. brasiliensis treated with compounds. DETAILED DESCRIPTION
[0023] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.
[0024] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.
[0025] The compounds used in the examples were purchased from Taoshu Biotechnology Co., Ltd., with serial number E002-0872 and catalog number IBS-E0356732; BALB / c mice were purchased from Guangdong Medical Laboratory Animal Center; Nippostrongylus brasiliensis was obtained from South China Agricultural University; and MTT reagent was purchased from Solebol.
[0026] Example 1 Inhibition rate of compound on egg hatching 1. Effects of compounds on the hatching of N. brasiliensis eggs The egg hatching inhibition experiment was conducted using compounds at concentrations of 10 μM, 20 μM, 40 μM, and 80 μM, respectively. The specific method is as follows: (1) On the 11th day after infection, the positive SD rats were euthanized, the small intestine was removed and longitudinally dissected, and the female adult worms in the process of laying eggs were picked out under a stereomicroscope; (2) Wash the adult worms three times with PBS, place them in culture medium, incubate at 37°C for 1 hour, and wait for the adult worms to lay eggs. Remove the adult worms from the culture dish, collect the eggs, transfer them to a 96-well cell culture plate, and count the eggs. (3) Add the compound to a 96-well cell culture plate containing parasite eggs to a final working concentration of 10 μM, 20 μM, 40 μM, and 80 μM. Place the culture plate in an incubator at 37°C and 5% CO2, and observe the hatching of the parasite eggs under an inverted microscope every 6 hours. After 16 hours, count the L1 larvae that have hatched in each well. The ratio of the number of L1 larvae to the number of parasite eggs in each well is calculated to determine the egg hatching rate.
[0027] The statistical results of the compounds on the hatching rate of Nippostrongylus brasiliensis eggs are as follows Figure 1 As shown in Table 1, the compound can inhibit the hatching of N. brasiliensis eggs. At a concentration of 40 μM, the inhibitory effect on egg hatching is significantly higher than that of the positive control albendazole, and at a concentration of 80 μM, it can achieve 100% inhibition, showing a significant egg hatching inhibitory effect.
[0028] Table 1 Egg hatching rate at different compound concentrations
[0029] 2. Cell proliferation inhibition experiment MTT reagent was used to determine the effect of the compound on the growth of HEK-293 cells at a concentration of 10 μM. HEK-293T cells were digested, counted and aliquoted into 96-well cell culture plates. After culture for 24 hours, the culture medium was discarded and replaced with serum-free culture medium to which the compound had been added. The specific groups included: albendazole positive control group, DMSO negative control group, and compound group. The cells were placed in a cell culture incubator and cultured for 24 hours; the culture medium was discarded and replaced with 200 μL serum-free culture medium per well. 20 μL of MTT colorimetric reagent was added to each well, followed by incubation at 37°C for 1 to 3 hours. After incubation, the plate was placed in a microplate reader and the OD was read. 490 Fluorescence value.
[0030] The results showed that the inhibition rate of the positive control albendazole on the cell proliferation of HEK-293T cells was 28.4%, and the inhibition rate of the compound on the cell proliferation of HEK-293T cells was 25.5%, and its effect on cell proliferation was less than that of the positive control.
[0031] Example 2 In vivo insecticide reduction effect of the compound The compound was used at a dose of 15 mg / kg to assess its in vivo worm-reducing effect in BALB / c mice infected with N. brasiliensis. Mice were inoculated with 800 L3 larvae per mouse. The 15 mg / kg dose of the compound served as the experimental group, while 20 mg / kg of albendazole served as the positive control. A solvent of 10% DMSO, 40% PEG-300, and 50% saline was used as the solvent; the negative control received an equal volume of solvent. The first treatment was administered 2 hours after infection, followed by daily intraperitoneal injections for 3 days. Mice were dissected on day 8 after infection, and the intestinal worm load was recorded. The specific steps were as follows: (1) Six-week-old female BALB / c mice were infected with N. brasiliensis subcutaneously via the neck with 800 L3 larvae / mouse as the infection dose using PBS as the diluent, with 4 mice in each group; (2) On the 8th day after infection, the mice were euthanized and dissected, and the number of worms in the mice's intestines was calculated under a stereomicroscope.
[0032] The percentage of worms collected from the intestines of mice in each group was calculated by taking the worm load in the negative control group as the control. The results are as follows: Figure 2 As shown, the compound significantly reduced the number of Brazilian roundworms in the intestines of infected mice, with a worm reduction rate of 61.7%, and had an anti-Brazilian roundworm infection effect.
[0033] Example 3 Effect of the Compound on Reducing EPG (Eggs per Gram of Feces) in Infected Mice The experimental group was assigned to the compound at a dose of 15 mg / kg for the measurement of EPG in the feces of infected mice. BALB / c mice were inoculated with 800 L3 larvae of N. brasiliensis. The experimental group received the compound at a dose of 15 mg / kg, while the positive control group received 20 mg / kg albendazole. The solvent was 10% DMSO, 40% PEG-300, and 50% saline. The negative control group received an equal volume of solvent. The first treatment was administered 2 hours after infection, followed by daily intraperitoneal injections for 3 days. EPG was measured on day 8 after infection. The specific steps were as follows: (1) Six-week-old female BALB / c mice were infected with N. brasiliensis subcutaneously via the neck with 800 L3 larvae per mouse as the infection dose using PBS as the diluent. Four mice were inoculated per group. (2) On the 8th day after infection, collect the fresh feces of the infected mice and weigh them. Add pure water until the feces are just submerged. Wait for 5 minutes until the feces become soft. After crushing, transfer the fecal suspension to a 2 mL centrifuge tube, add an appropriate amount of saturated salt water, mix thoroughly, and draw the suspension into the counting chamber of the McMaster plate with a Pasteur tube. After standing for 5 minutes, use an ordinary optical microscope to examine and count.
[0034] The counting results are as follows Figure 3 As shown, the compound can significantly reduce the number of N. brasiliensis eggs in the feces of infected mice and has an anti-N. brasiliensis infection effect.
[0035] Example 4 Effect of the Compound on Improving Intestinal Lesions in Infected Mice The compound was administered at a dose of 15 mg / kg to measure intestinal pathology in infected mice. BALB / c mice infected with N. brasiliensis were inoculated with 800 L3 larvae per mouse. Two hours later, the first treatment (15 mg / kg compound, with 20 mg / kg albendazole as a positive control, and the compound was administered in a solution of 10% DMSO, 40% PEG300, and 50% saline; the negative control was administered with an equal volume of 10% DMSO, 40% PEG300, and 50% saline) was administered. Treatments were administered intraperitoneally daily for three days. On day eight post-infection, the mice were dissected to remove the intestines and measure the ratio of villus length to crypt depth. The specific steps were as follows: (1) PBS buffer was used as the diluent and 800 L3 larvae / mouse were used as the infection dose. Six-week-old female BALB / c mice were infected with N. brasiliensis subcutaneously at the back of the neck, with 4 mice in each group. (2) On the 8th day after infection, the mice were dissected and the duodenal segments were removed. After simple cleaning, they were quickly placed in 4% paraformaldehyde fixative. (3) After fixation at room temperature for 48 hours, rinse off the surface paraformaldehyde, dehydrate with ethanol gradient, and then make it transparent with xylene for 2 hours. After being immersed in wax and embedded, slice the slices with a paraffin slicer. The prepared paraffin slices are dewaxed with xylene, gradually dehydrated with ethanol, stained with hematoxylin, differentiated with hydrochloric acid and ethanol, and then stained with eosin; dehydrate with ethanol gradient, make the tissue fully transparent with xylene, seal the slices with neutral gum, observe under a microscope and take pictures; (4) Use Image J to measure the length of intestinal villi and the depth of their crypts, and finally calculate their ratio.
[0036] The ratio results are as follows Figure 4 As shown, when treated with a dose of 15 mg / kg, the compound can significantly improve the ratio of intestinal villus length to crypt depth in infected mice, reduce the degree of lesions in the parasitic site of the Brazilian roundworm, and can be used to reduce or alleviate the symptoms of Brazilian roundworm infection.
[0037] In summary, the present invention provides the use of compounds for combating N. braziliani infection and inhibiting egg hatching. By screening compounds that target the FAR-1 protein of N. braziliani, the present invention developed compounds with excellent anti-parasitic effects that can significantly inhibit the hatching of N. braziliani eggs. Furthermore, the compounds can inhibit the growth and development of N. braziliani nematodes in mice, demonstrating significant anti-nematode infection effects and resistance to N. braziliani infection. Furthermore, the compounds have good biosafety, exhibiting minimal cell proliferation inhibition, and exhibit low mouse mortality and low toxicity to mice. These compounds provide more effective drugs for combating nematode infections and can be used to prepare and develop more products for inhibiting nematode growth and drugs for combating nematode infections, providing methods and products for developing more drugs for combating nematode infections.
[0038] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.
Claims
1. Use of a compound in the preparation of a drug for resisting nematode infection, characterized in that: The structural formula of the compound is shown below:
2. Use of a compound in inhibiting nematode growth, characterized in that: The structural formula of the compound is shown below:
3. Use of a compound in the preparation of a product for inhibiting the growth and development of nematodes, characterized in that: The structural formula of the compound is shown below:
4. Use of a compound in inhibiting the hatching of nematode eggs, characterized in that: The structural formula of the compound is shown below:
5. Use of a compound in the preparation of a product for inhibiting the hatching of nematode eggs, characterized in that: The structural formula of the compound is shown below:
6. Use of a compound in the preparation of a drug for alleviating pathological changes in parasitic sites of nematodes, characterized in that: The structural formula of the compound is shown below:
7. The use according to any one of claims 1 to 6, characterized in that: The nematode is Nipostrongylus brasiliensis.
8. The application according to claim 1, characterized in that: The drug can reduce the number of nematodes.
9. The use according to claim 1, characterized in that: The drug can reduce the number of nematode eggs.
10. The application according to claim 1 or 6, characterized in that: The medicine further includes a pharmaceutically acceptable carrier or excipient.