Application of compound in preparation of medicine for resisting nematode infection

Abstract

The invention discloses an application of a compound in preparation of a medicine for resisting nematode infection. The invention provides a novel application of a compound in resisting nematode infection, and researches show that the compound # imgabs0 can inhibit nematode egg incubation, has better safety than a positive drug albendazole, and can significantly inhibit growth and reproduction of nematodes. In a mouse body infected with nematodes, the compound can significantly reduce the number of Brazilian strongylus, reduce the number of eggs in host excrement, effectively inhibit the reproduction of Brazilian strongylus and inhibit the growth of Angiostrongylus cantonensis, has a significant nematode infection resistance effect, does not affect the growth of the mouse, and has a good application prospect. The compound can be used for preparing and developing more products for resisting nematode infection and medicines for treating diseases caused by nematode infection, and has important significance on research and development of medicines for preventing and controlling nematode infection.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to the use of a compound in the preparation of a drug for resisting nematode infection. Background Art

[0002] Nematode infections can affect human health. For example, intestinal parasites like roundworms can lead to protein malabsorption in children, impacting growth and development. Hookworms can cause blood-sucking anemia in humans and even heart failure. Nematode infections in wildlife can also severely impact the health and conservation of rare species. Diarrhea caused by Trichocera infestation is a significant threat to the conservation of Sichuan golden snub-nosed monkeys. Nematodes can also infect a wide range of farmed animals, including pigs, cattle, and sheep, causing declines in meat production, undermining the livestock industry and resulting in significant economic losses.

[0003] Japanese roundworm (Nipponstrongylus brasiliensis) is a soil-borne intestinal parasitic nematode that infects rodents percutaneously or orally. Its life cycle consists of free-living and internal parasitic stages. Infectious L3 larvae enter the internal parasitic stage after percutaneous infection of mice. They develop through the respiratory and digestive tracts, eventually developing into dioecious adults in the small intestine and beginning to excrete eggs six days after infection. The zoonotic Angiostrongylus cantonensis is a foodborne parasitic nematode that requires both intermediate and definitive hosts to complete its complex life cycle. Its non-suitable hosts are mice and various mammals. In non-suitable hosts, the worm's development arrests at the brain parasitic stage, causing severe damage and pathology to the host's brain.

[0004] Currently, the range of drugs used to control nematode infections is very limited. Mainstream anthelmintics include benzimidazoles (such as albendazole), macrolides (ivermectin), and imidazothiazoles (levamisole). Although broad-spectrum, long-term, irregular use has already led to drug resistance. Regarding vaccines and other biological agents, only one commercially available vaccine, Barbervax, is available for injection against Haemonchus contortus in sheep. Therefore, the development of new, highly effective, and low-toxic anti-nematode drugs remains of vital importance. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of existing anti-nematode drugs, such as large side effects and low safety, and provide an application of a compound in the preparation of drugs for anti-nematode infection.

[0006] The purpose of the present invention is to provide a new application of a compound in resisting nematode infection.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions: In the early stages of this study, a virtual screening of compounds targeting the nematode FAR-1 protein revealed a compound that binds to the FAR-1 protein. This compound effectively inhibits nematode growth and development, demonstrating excellent anti-nematode efficacy. In vitro experiments demonstrated that the compound inhibited the hatching of N. brasiliensis eggs and had minimal inhibitory activity on HEK-293T cell proliferation, less than the positively labeled drug albendazole, demonstrating a good safety profile. In vivo experiments demonstrated that the compound inhibited N. brasiliensis growth in mice, reduced the number of N. brasiliensis worms in the mouse intestines, and significantly reduced the number of eggs excreted in the feces of infected mice, inhibiting N. brasiliensis worm reproduction. Furthermore, the compound improved intestinal lesions in mice infected with the nematode, demonstrating a moderate protective effect against N. brasiliensis infection. Furthermore, studies have shown that the compound also effectively reduced the number of Angiostrongylus cantonensis worms in the mouse brain, demonstrating a moderate protective effect against A. cantonensis infection in mice. The present invention provides a new application of the compound in resisting nematode infection, provides new ideas and methods for preparing and developing more anti-nematode drugs, and has good application prospects in the research and development of drugs for preparing, treating or preventing nematode infections.

[0008] Furthermore, the English name of the compound is 5-(3-bromophenyl)-4-[(4-fluorophenyl)carbonyl]-3-hydroxy-1-[3-(1H-imidazol-1-yl)propyl]-1,5-dihydro-2H-pyrrol-2-one, the CAS number is 381180-44-5, and its structural formula is:

[0009] Therefore, the present invention provides use of the compound in preparing a drug for resisting nematode infection.

[0010] The present invention provides application of a compound in inhibiting the growth and reproduction of nematodes.

[0011] The present invention provides use of a compound in inhibiting the hatching of nematode eggs.

[0012] The present invention provides use of a compound in preparing a product for inhibiting the hatching of nematode eggs.

[0013] The present invention provides use of a compound in preparing a medicine for alleviating / relieving pathological changes after nematode infection.

[0014] Preferably, the nematode is N. brasiliensis or Angiostrongylus cantonensis.

[0015] Furthermore, the drug can reduce the number of nematodes.

[0016] Furthermore, the drug can reduce the number of nematode eggs.

[0017] Furthermore, the drug further comprises pharmaceutically acceptable excipients or adjuvants.

[0018] Preferably, the dosage form of the drug is tablet, capsule, oral liquid preparation, spray or injection.

[0019] The present invention has the following beneficial effects: The present invention provides novel applications of compounds for combating nematode infections. Studies have shown that the compounds can inhibit the hatching of Nippostrongylus brasiliensis eggs and exhibit good cellular safety, with their inhibitory activity against cell proliferation lower than that of the positive drug albendazole. Furthermore, the compounds provided by the present invention can inhibit the growth and development of nematodes in mice, combating nematode infection without affecting mouse growth. These compounds exhibit good efficacy and anti-nematode potential, and can be used to prepare and develop additional anti-nematode products and drugs for treating nematode infections, demonstrating promising application prospects for the development of drugs for treating or preventing nematode infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The graph shows the effect of the compounds on the hatching of Nippostrongylus brasiliensis eggs (Note: the compound No. 4340-0245 in the figure is the compound, the same below).

[0021] Figure 2 Figure 2 shows the number of N. brasiliensis in mice after compound treatment.

[0022] Figure 3 This figure shows the number of eggs excreted by N. brasiliensis in mice after treatment with the compound.

[0023] Figure 4 Figure 2 is the ratio of intestinal villus height to crypt depth in mice infected with compound-treated N. brasiliensis.

[0024] Figure 5 Figure 2 is a graph showing the number of Angiostrongylus cantonensis in mice treated with the compound.

[0025] Figure 6 The graph shows the survival of mice treated with the compound. DETAILED DESCRIPTION

[0026] 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.

[0027] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0028] Example 1 Inhibition rate of compound on egg hatching 1. Effects of compounds on the hatching of N. brasiliensis eggs The compound (purchased from Taoshu Biotechnology Co., Ltd., No. 4340-0245, Catalog No. IBS-E0095393) at concentrations of 10 μM, 20 μM, 40 μM, and 80 μM was used to perform an egg hatching inhibition experiment with albendazole as a positive control. The specific method is as follows: (1) On the 11th day after infection, the positive SD rats were euthanized and the small intestine was removed and longitudinally dissected. The female adult worms in the process of laying eggs were picked out under a stereomicroscope.

[0029] (2) Wash the adult worms three times with PBS, place them in culture medium, and incubate them at 37°C for 1 hour to 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.

[0030] (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.

[0031] 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 results showed that the compound had a lower egg hatching rate than the positive control at concentrations of 40 μM and 80 μM, and achieved a 100% inhibitory effect at a concentration of 80 μM, significantly inhibiting the hatching of N. brasiliensis eggs.

[0032] Table 1 Egg hatching rate of compounds at different concentrations

[0033] 2. Cell proliferation inhibition experiment The inhibitory effect of the compound on cell proliferation was determined using MTT reagent (purchased from Solebro) to determine the effect of the compound at a concentration of 10 μM on the growth of HEK-293 cells. The specific steps are as follows: HEK-293T cells were digested, counted, and aliquoted into 96-well cell culture plates. Culture was performed for 24 hours. The culture medium was discarded and replaced with serum-free medium supplemented with the compound. Groups were divided into: albendazole positive control group, DMSO negative control group, and compound group. Culture was continued in a cell culture incubator for 24 hours. The culture medium was discarded and replaced with 200 μL of serum-free medium per well. 20 μL of MTT colorimetric reagent was added to each well, followed by incubation at 37°C for 1–3 hours. After incubation, the cells were placed in a microplate reader and the fluorescence value at OD490 was read.

[0034] The results showed that the cell proliferation inhibition rate of HEK-293T cells treated with 10 μM positive control albendazole was 28.4%, while the cell proliferation inhibition rate of HEK-293T cells treated with 10 μM compound was 7.8%, indicating that the compound had less inhibitory effect on cell proliferation than cells treated with the positive control albendazole and had better cell safety than albendazole.

[0035] Example 2 In vivo worm-reducing effect of the compound on Nipostrongylus brasiliensis The compounds were administered at a dose of 15 mg / kg to assess their in vivo worm-reducing effects in BALB / c mice (purchased from the Guangdong Medical Laboratory Animal Center) infected with N. brasiliensis. Mice were inoculated with 800 L3 larvae of N. brasiliensis (from South China Agricultural University). Two hours later, the first treatment (15 mg / kg of the compound as the experimental group, 20 mg / kg of albendazole as the positive control, and the compound was administered in a solution of 10% DMSO + 40% PEG300 + 50% saline; the negative control was administered with an equal volume of 10% DMSO + 40% PEG300 + 50% saline) was administered daily for three days. Mice were dissected on day eight post-infection, and the intestinal worm load was recorded. The specific steps were as follows: (1) Using PBS buffer as the diluent, mice were inoculated with 800 L3 larvae per mouse as the infection dose. Six-week-old female BALB / c mice were infected with N. brasiliensis by subcutaneous injection at the back of the neck, with 4 mice in each group.

[0036] (2) On the 8th day after infection, the mice were dissected and the number of worms in their intestines was calculated under a stereomicroscope.

[0037] 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 can reduce the number of Brazilian roundworms in the intestines of infected mice. The compound has a significant worm reduction effect when treated with a dose of 15 mg / kg, with a worm reduction rate of 62.2%, which can achieve resistance to Brazilian roundworm infection.

[0038] Example 3 Effects of Compounds on EPG-Infected Mice The EPG (egg count per gram of feces) of infected mice was measured using a 15 mg / kg dose of the compound. BALB / c mice (purchased from the Guangdong Medical Laboratory Animal Center) infected with N. brasiliensis (from South China Agricultural University) were inoculated with 800 L3 larvae per mouse. Two hours later, the first treatment (15 mg / kg of the compound as the experimental group, 20 mg / kg of albendazole as the positive control, and the compound was treated with 10% DMSO + 40% PEG300 + 50% saline as the solvent; the negative control was given an equal volume of 10% DMSO + 40% PEG300 + 50% saline)) was administered intraperitoneally daily for three days. EPG levels in infected mice were measured on day eight post-infection. The specific steps were as follows: (1) Using PBS buffer as the diluent, mice were inoculated with 800 L3 larvae per mouse as the infection dose. Six-week-old female BALB / c mice were infected with N. brasiliensis by subcutaneous injection at the back of the neck, with 4 mice in each group.

[0039] (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 egg counting plate through a Pasteur tube. After standing for 5 minutes, use an ordinary optical microscope to examine and count the eggs.

[0040] The counting results are as follows Figure 3 As shown, when the compound is treated with a dose of 15 mg / kg, the number of eggs discharged by mice infected with the Japanese strongyloidiasis can be significantly reduced, and the compound has an anti-Japanese strongyloidiasis infection effect.

[0041] Example 4 Effects of Compounds on Intestinal Pathology in Infected Mice The compound was administered at a 15 mg / kg dose to measure intestinal pathology in infected mice. BALB / c mice (purchased from the Guangdong Medical Laboratory Animal Center) infected with N. brasiliensis (sourced from South China Agricultural University) were inoculated with 800 L3 larvae per mouse. Two hours later, the first treatment (15 mg / kg compound as the experimental group, 20 mg / kg albendazole as the 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 intraperitoneally injected daily for three days. On day eight post-infection, the mice were dissected to remove the intestines. Intestinal pathological changes were observed by H&E staining, and the ratio of intestinal villus to crypt depth was measured. The specific steps were as follows: (1) Using PBS buffer as the diluent, mice were inoculated with 800 L3 larvae / mouse as the infection dose. Six-week-old female BALB / c mice were infected with N. brasiliensis by subcutaneous injection at the back of the neck, with 4 mice in each group.

[0042] (2) On the 8th day after infection, the mice were dissected and the duodenal segments were cut out. After simple cleaning, they were quickly placed in 4% paraformaldehyde fixative.

[0043] (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 using a paraffin slicer. The prepared paraffin sections are dewaxed with xylene, gradually dehydrated with ethanol, stained with hematoxylin, differentiated with hydrochloric acid and ethanol, and then stained with eosin. After dehydration with ethanol gradient, the tissue is fully transparent with xylene, and then the sections are sealed with neutral gum, observed under a microscope, and photographed.

[0044] (4) Use Image J to measure the length of intestinal villi and the depth of their crypts, and finally calculate their ratio.

[0045] The results are as follows Figure 4 As shown, when treated with the compound at a dose of 15 mg / kg, the ratio of intestinal villus length to crypt depth in infected mice was significantly improved, the degree of intestinal lesions in mice was reduced, and the symptoms after nematode infection were alleviated or relieved.

[0046] Example 5 The compound's worm-reducing effect on Angiostrongylus cantonensis In vivo worm reduction assays were performed using a 15 mg / kg dose of the compound in BALB / c mice infected with Angiostrongylus cantonensis (purchased from the Guangdong Medical Laboratory Animal Center). Mice were inoculated with 50 L3 larvae of Angiostrongylus cantonensis (from South China Agricultural University) per mouse. The first treatment was administered 24 hours later (15 mg / kg of the compound as the experimental group, 20 mg / kg of albendazole as the 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). Treatments were administered intraperitoneally daily for 7 days. On day 16 post-infection, mice were dissected and the number of L4 larvae in brain tissue was recorded. The specific steps were as follows: (1) Six-week-old male BALB / c mice were inoculated with Angiostrongylus cantonensis by gavage using PBS buffer as the diluent and 50 L3 larvae / mouse as the infection dose. Four mice were inoculated in each group.

[0047] (2) Mice were dissected on the 15th day of infection, and the number of L4 larvae of Angiostrongylus cantonensis in the mouse brain tissue was counted under a stereomicroscope.

[0048] The counting results are as follows Figure 5 As shown, the compound can significantly reduce the number of Guangzhou roundworms in the brain tissue of infected mice. When treated with a dose of 15 mg / kg, the worm reduction rate of infected mice reached 52.7%, which is able to resist Guangzhou roundworm infection.

[0049] Example 6 Toxicity test of compounds on mice In vivo toxicity testing was performed on BALB / c mice (purchased from the Guangdong Medical Laboratory Animal Center) using a 50 mg / kg dose of the compound. Using 10% DMSO + 40% PEG300 + 50% saline as the solvent, the experimental group received a 50 mg / kg dose, while the control group received an equal volume of 10% DMSO + 40% PEG300 + 50% saline. Mice were observed for mortality and mental status for 14 days after a single intraperitoneal injection. The specific steps were as follows: (1) Six female and six male BALB / c mice were selected and divided into four groups (two males and two females) after adaptive feeding. They were fasted but not watered for 12 hours before the experiment. After fasting, each mouse in each group was marked.

[0050] (2) Immediately after intraperitoneal injection, mice were observed for vital signs at 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 14 days after injection. Observations included survival and abnormal behavioral states such as convulsions, lethargy, lethargy, and hair loss.

[0051] The survival results of mice were as follows Figure 6 As shown, no mice in the compound group died during the experimental period, their activity status showed no significant difference compared with the control group, and no obvious toxic reaction symptoms such as convulsions, breathing difficulties or movement disorders were observed, indicating that the compound is relatively safe.

[0052] 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 the growth and reproduction of nematodes, characterized in that: The structural formula of the compound is shown below:

3. Use of a compound in inhibiting the hatching of nematode eggs, characterized in that: The structural formula of the compound is shown below:

4. 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:

5. Use of a compound in the preparation of a drug for alleviating / relieving lesions after nematode infection, characterized in that: The structural formula of the compound is shown below:

6. The use according to any one of claims 1 to 5, characterized in that: The nematode is N. brasiliensis or Angiostrongylus cantonensis.

7. The use according to claim 1 or 5, characterized in that: The drug can reduce the number of nematodes.

8. The use according to claim 1 or 5, characterized in that: The drug can reduce the number of nematode eggs.

9. The application according to claim 1 or 5, characterized in that The drug further comprises pharmaceutically acceptable excipients or adjuvants.

10. The application according to claim 1 or 5, characterized in that: The dosage form of the drug is tablet, capsule, oral liquid preparation, spray or injection.

Images