Application of boscalid in preparation of medicine for treating toxoplasmosis

Abstract

The invention provides application of boscalid in preparation of a medicine for treating toxoplasmosis, and particularly relates to application of boscalid in preparation of a medicine for preventing or treating toxoplasmosis infection and a composition for preventing or treating toxoplasmosis infection, and the active ingredient of the composition comprises boscalid. The inventor finds and verifies that the boscalid has a remarkable inhibitory activity effect on intracellular parasitic protozoa toxoplasma gondii through a large number of researches and experiments, Vero cells infected with the toxoplasma gondii are used as cells of an experimental model, and EC50 determination shows that the boscalid has half of the inhibitory effect on the toxoplasma gondii when the application amount of the boscalid is 40.19 mu M. Meanwhile, when the application amount of the boscalid is 10000 [mu] M, the boscalid still has no cytotoxicity to Vero cells. Compared with existing drugs for treating toxoplasma gondii infection, the effective inhibition concentration of the boscalid provided by the invention is far smaller than the cytotoxicity of the boscalid, and the boscalid has the remarkable advantages of high efficiency, low toxicity and safety when being used for preparing the drugs for preventing or treating toxoplasma gondii infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of parasitic disease prevention and treatment, and relates to the use of boscalid in preventing and treating Toxoplasma gondii infection, and specifically relates to the use of boscalid in preparing a medicament for preventing or treating Toxoplasma gondii infection, and a composition for preventing or treating Toxoplasma gondii infection. Background Art

[0002] Toxoplasmosis is an obligate intracellular parasitic protozoan disease caused by Toxoplasma gondii. Toxoplasma gondii infects almost all warm-blooded animals and humans, even some cold-blooded animals, and can parasitize all nucleated cells in the animal body. According to statistics, about one-third of the world's population is infected with Toxoplasma gondii, and the serum positivity rate of Toxoplasma gondii varies from region to region. For example, the positivity rate in the Americas, Europe and Asia is about 30%, while the positivity rate in Africa is over 60%. Most people have latent toxoplasmosis infection, but infants and immunosuppressed patients (such as AIDS patients, organ transplant patients and malignant tumor patients) infected with Toxoplasma gondii will develop severe or fatal diseases. Toxoplasmosis infection is also an important cause of miscarriage, stillbirth and other reproductive disorders in pregnant animals and pregnant women, and is one of the must-check items for prenatal examinations in my country.

[0003] In the livestock sector, a wide range of animals (pigs, cattle, sheep, horses, dogs, cats, chickens, etc.) have high infection rates for Toxoplasma gondii. Serological surveys indicate that up to 30% of pigs worldwide are infected with Toxoplasma gondii, and in parts of my country, infection rates in pigs reach over 70%. When acute toxoplasmosis occurs on pig farms, the morbidity rate can reach 100%, with mortality rates exceeding 60%. In the United States and China, pigs are considered the most important transmitters of Toxoplasma among food animals, posing a significant public health and safety risk. my country ranks first in the world in pork production, and an outbreak of Toxoplasmosis in pigs would result in significant economic losses to the livestock industry and potentially become a serious food safety issue.

[0004] Currently, clinical treatment for toxoplasmosis still relies on chemical drugs. Due to the complexity of the parasite's life cycle, diverse pathogenesis, and variability in its biological characteristics, there are currently no preventive or specific drug treatments. Although the combination of pyrimethamine and sulfadiazine is currently the gold standard for clinical treatment of toxoplasmosis, this treatment method is often accompanied by serious side effects, is incomplete, and is prone to relapse, resulting in a high failure rate. Therefore, screening for safe and effective anti-toxoplasmosis drugs is an urgent issue and has great market prospects.

[0005] In summary, developing a drug that can effectively prevent or treat Toxoplasma gondii infection is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0006] In view of the problems in the prior art of drugs for preventing or treating Toxoplasma infection, such as the tendency to induce drug resistance, significant toxic side effects, and poor efficacy in preventing and treating Toxoplasma infection due to high drug dosage, the present invention specifically provides the use of boscalid in the preparation of drugs for preventing or treating Toxoplasma infection, which specifically includes:

[0007] One of the purposes of the present invention is to provide the use of boscalid in the preparation of a drug for preventing or treating toxoplasma gondii infection. The CAS number of boscalid is 188425-85-6, and its molecular formula is C 18 H 12 Cl2N2O, chemical formula is:

[0008]

[0009] Boscalid is a new nicotinamide fungicide developed by BASF of Germany. It is a succinate dehydrogenase inhibitor (SDHI) and plays a key role in the prevention and control of plant diseases.

[0010] Its reaction mechanism primarily affects the pathogen's mitochondrial respiratory chain: when boscalid acts on plant pathogens, it specifically inhibits succinate-CoA reductase, an enzyme in the mitochondrial electron transport chain. In the tricarboxylic acid cycle, succinate dehydrogenase catalyzes the conversion of succinate to fumarate. Boscalid's inhibition of this enzyme blocks the normal functioning of the tricarboxylic acid cycle (a crucial pathway for cellular energy production), leading to a deficiency of amino acids and sugars within the pathogen's cells. This, in turn, interferes with the pathogen's cell division and growth, preventing normal reproduction and plant infestation, ultimately achieving the goal of controlling the disease. Furthermore, boscalid affects the entire growth chain of the pathogen, with a particularly significant effect in inhibiting spore germination. Even at low concentrations, it can hinder hyphal growth and spore formation, providing both protective and therapeutic benefits, effectively preventing secondary infections after the onset of disease.

[0011] Due to its significant effectiveness against a variety of fungal diseases, including powdery mildew, gray mold, and sclerotinia, boscalid is now widely used in crops such as grapes, strawberries, cucumbers, and tomatoes. Furthermore, the environmentally friendly agent has been registered in major agricultural production areas both domestically and internationally, making it a key tool for controlling fungal diseases in modern plant protection.

[0012] In a preferred embodiment, the drug for preventing or treating Toxoplasma gondii infection is a drug for preventing or treating toxoplasmosis.

[0013] In a preferred embodiment, the drug for preventing or treating Toxoplasma gondii infection is a drug that inhibits the proliferation of Toxoplasma gondii or inhibits the ability of Toxoplasma gondii to lyse cells.

[0014] In a preferred embodiment, the drug for preventing or treating Toxoplasma gondii infection is a drug for eliminating Toxoplasma gondii in humans or non-human animals, including but not limited to pigs, cattle, sheep, horses, dogs, cats, chickens, etc., which are susceptible to Toxoplasma gondii.

[0015] In a preferred embodiment, by measuring EC 50 When the application amount of boscalid is 40.19 μM, it has a half inhibitory effect on Toxoplasma gondii.

[0016] In a preferred embodiment, when the amount of boscalid applied is 10000 μM, it has no cytotoxicity to Vero cells.

[0017] Another object of the present invention is to provide a composition for preventing or treating Toxoplasma gondii infection, wherein the active ingredient of the composition includes boscalid.

[0018] In a preferred embodiment, the composition further comprises one or more of a diluent, a wetting agent, a binder, a disintegrant, a lubricant, a color and flavor regulator, a solvent, a solubilizer, a cosolvent, an emulsifier, an antioxidant, a metal complexing agent, a preservative, a pH regulator, a surfactant, an excipient, a filler and a synergist.

[0019] In a preferred embodiment, the diluent includes starch, sucrose, cellulose, inorganic salts, etc.; the wetting agent includes water, ethanol, etc.; the binder includes starch slurry, dextrin, sugar, cellulose derivatives, gelatin, povidone, polyethylene glycol, etc.; the disintegrant includes starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, sodium dicarboxymethyl cellulose, surfactants, etc.; the lubricant includes talc, calcium stearate, magnesium stearate, magnesium lauryl sulfate, polyethylene glycol, etc.; the color, flavor and flavoring agent includes pigment, sweetener, flavor, mucilage, etc.; the solvent includes water, glycerol, ethanol, etc.; the solubilizer includes Tweens, sorbits, sulfates, sulfonates, etc.; the cosolvents include organic acids (such as citric acid) and their salts, inorganic salts, polyethylene glycol, etc.; the emulsifiers include spans, glycerol fatty acid esters, gum arabic, gelatin, agar, sodium alginate, etc.; the antioxidants include sulfites, ascorbic acid, gallic acid and its salts, etc.; the metal chelating agents include disodium edetate, polycarboxylic acid compounds, etc.; the preservatives include parabens, quaternary ammonium compounds, chlorhexidine acetate, etc.; the pH adjusters include hydrochloric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphates, citrates, etc.

[0020] In the present invention, the composition dosage forms include, but are not limited to, powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, and other oral dosage forms, topical preparations, suppositories, and sterile injectable solutions. It will be appreciated that the boscalid-containing composition of the present invention can be administered in a variety of ways, depending on the different excipients and dosage forms.

[0021] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0022] 1. Conventional anti-Toxoplasma drugs in the prior art, such as pyrimethamine and sulfadiazine, work by inhibiting Toxoplasma gondii's dihydrofolate reductase and dihydrofolate synthase, respectively, thereby interfering with folate metabolism and producing an anti-Toxoplasma effect. However, with long-term use, both pyrimethamine and sulfadiazine carry a certain risk of cytotoxicity and are also prone to inducing drug resistance in parasite strains. In light of this, the present inventors, through extensive research, have discovered that boscalid has significant inhibitory activity against the intracellular parasite Toxoplasma gondii, with minimal toxic side effects. This not only provides a new drug for combating Toxoplasma infection, but also expands new avenues for the effective utilization of boscalid.

[0023] 2. The present inventors have verified through a large number of experiments that boscalid has a significant effect in preventing or treating Toxoplasma gondii infection. Specifically, using Vero cells infected with Toxoplasma gondii as experimental models, the EC 50 The results showed that boscalid had a 50% inhibitory effect on Toxoplasma gondii at 40.19 μM. Therefore, a small amount of application can achieve a highly effective anti-Toxoplasma infection effect.

[0024] 3. Furthermore, experiments have confirmed that boscalid, as an anti-Toxoplasma drug, has the advantage of low toxicity. Specifically, the cytotoxicity of boscalid to Vero cells was tested using the CCK8 assay. The results showed that even at a maximum dose of 10,000 μM, boscalid remained non-cytotoxic to Vero cells. Therefore, compared to existing drugs for treating Toxoplasma infection, which suffer from significant side effects, the effective inhibitory concentration of boscalid provided in the present invention is far lower than its cytotoxicity. Therefore, its use in the preparation of drugs for the prevention or treatment of Toxoplasma infection offers the significant advantages of high efficacy, low toxicity, and safety.

[0025] 4. The present invention has found through a large number of experimental explorations that the anti-Toxoplasma effect of boscalid is mainly through inhibiting its intracellular proliferation, and the effect of boscalid on Toxoplasma is dose-dependent, indicating that the higher the concentration, the better the anti-insect effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, so that the advantages of the present invention become clearer and easier to understand, wherein:

[0027] Figure 1 This is a graph showing the results of a toxicity test on Vero cells in Example 1 of the present invention;

[0028] Figure 2 This is a graph showing the inhibitory effect of boscalid on Toxoplasma gondii in Example 2 of the present invention;

[0029] Figure 3 This is a plaque test diagram of boscalid and a control group in Example 3 of the present invention;

[0030] Figure 4 This is a statistical graph showing the anti-proliferative effects of boscalid and the control group on intracellular RH tachyzoites in Example 4 of the present invention;

[0031] Figure 5 This is a graph showing the anti-invasion effect of boscalid and the control group on extracellular RH-type Toxoplasma gondii in Example 4 of the present invention. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments. However, it should be understood that the protection scope of the present invention is not limited to the specific embodiments.

[0033] The present invention provides the use of boscalid in the preparation of a drug for treating toxoplasmosis, thereby effectively solving the problems of existing anti-toxoplasmosis drugs (such as pyrimethamine and sulfadiazine) such as drug resistance easily caused by long-term use, severe toxic side effects, and poor effect in preventing and treating toxoplasmosis infection.

[0034] The present invention uses boscalid, which is conventionally used to eliminate plant fungal diseases, to eliminate Toxoplasma gondii in humans or non-human animals. This not only avoids the problem of drug resistance caused by long-term use of traditional anti-insect drugs, but also provides a highly effective, low-toxic and safe anti-Toxoplasma infection drug because the effective inhibitory concentration of boscalid on Toxoplasma is far lower than its cytotoxicity.

[0035] The technical solution of this application is described in detail below through specific embodiments:

[0036] Unless otherwise specified, the technical means used in the present invention are conventional means well known to those skilled in the art. The various raw materials, reagents, instruments, and equipment used in the present invention are commercially available or can be prepared by existing methods. Boscalid used in the present invention was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., and the Vero cells and HFF cells used in the present invention were purchased from the ATCC cell bank.

[0037] Example 1

[0038] Study on the toxicity of boscalid to Vero cells

[0039] 1. Experimental process

[0040] Cytotoxicity experiment: The CCK-8 method was used to determine the toxicity of boscalid to Vero cells. African green monkey kidney (Vero) cell suspension was inoculated in a 96-well cell culture plate, with approximately 100 μL (5000 Vero cells) per well. The culture plate was placed in an incubator for pre-culture for a period of time (37°C, 5% CO2) to allow the cells to adhere to the wall for about four hours. Nine concentration gradients were set from 40 μM to 10,000 μM. Vero cells were treated with different concentrations of boscalid to observe whether their cytotoxicity was significantly different from that of the 0.1% DMSO control group. The absorbance at 450 nm was measured using a microplate reader. The cell viability was calculated according to the following formula, and the independent experiment was repeated 3 times.

[0041] Cell viability (%) = (OD 实验组 -OD 空白组 ) / (OD 对照组 -OD 空白组)×100％

[0042] 2. Experimental results

[0043] like Figure 1 The absorbance values of African green monkey kidney cells treated with different concentrations of boscalid at 450nm were statistically analyzed. The results showed that there was no significant difference in cell absorbance between the boscalid-treated groups and the DMSO control group, indicating that boscalid has low toxicity and is safe for cells.

[0044] Example 2

[0045] Inhibitory Effects of Boscalid on Toxoplasma Gondii

[0046] 1. Experimental process

[0047] Vero cells were spread over 96-well cell culture plates, and fresh and vigorous Luciferase tachyzoites were harvested from the Vero cells. The tachyzoites were counted with a hemocytometer. 3×10 5 Tachyzoites were inoculated into Vero cells. DMSO was used as the control group, and different concentration gradients of boscalid from 0 to 500 μM were added. The culture medium in each well was controlled at 150-300 μL, and the independent experiment was repeated 3 times. After 24 hours of culture, the DMEM in the well was gently aspirated, and 100 μL of cell lysis buffer was added to each well. After lysis for 5 minutes, the lysate was aspirated, placed in a 1 mL EP tube, centrifuged at 11000 r / min for 5 minutes, 100 μL of supernatant was aspirated and added to a 96-well enzyme-labeled plate, and then 100 μL of luciferase was added to each well under dark conditions. After mixing, the luminescence value was detected by chemiluminescence instrument. The survival rate of luciferase was calculated.

[0048] Inhibition rate % = (RLU DMSO -RLU 处理组) / RLU DMSO ×100

[0049] 2. Experimental results

[0050] like Figure 2 The results showed that the half maximum effective concentration (EC50) of boscalid against Toxoplasma gondii 50 ) was 40.19 μM. Furthermore, the inhibitory effect on Toxoplasma gondii was dose-dependent, with higher concentrations increasing the inhibitory effect. This indicates that boscalid has a good inhibitory effect on Toxoplasma gondii.

[0051] Example 3

[0052] Effect of Boscalid on Inhibiting Plaque Formation of Intracellular Toxoplasma Gondii

[0053] 1. Experimental process

[0054] Human foreskin fibroblasts (HFF), a classic cell model for Toxoplasma research, were spread over 12-well cell culture plates, and tachyzoites of Toxoplasma type I strain (RH) were collected. Then, 150 tachyzoites were inoculated into a monolayer of HFF cells (the ratio of the number of inoculated Toxoplasma to the number of host cells was 1:2000). The cells were divided into two groups, of which 6 wells were added with an equal amount of DMSO as a control group, and the other 6 wells were added with 100 μM boscalid to each well and incubated for 7 days (the culture medium in each well was controlled at 2-3 mL). The plates were fixed with 4% paraformaldehyde at room temperature for 1 hour, washed with PBS, and stained with crystal violet to observe the size of the plaque area.

[0055] 2. Experimental results

[0056] like Figure 3 At the same magnification, larger plaques were observed in the DMSO negative control group, which was significantly different from the plaque area formed in the cells of the boscalid group, indicating that boscalid significantly inhibited the growth of Toxoplasma gondii.

[0057] Example 4

[0058] Antiproliferative Effects of Boscalid on Intracellular Toxoplasma Gondii Type I Strain (RH)

[0059] 1. Experimental process

[0060] Proliferation assay: 1×10 cells were seeded into each well of a 12-well flyer plate containing HFF cells in good growth condition. 5Fresh tachyzoites were inoculated at a ratio of 1:10 between the number of Toxoplasma gondii and the number of host cells. Two hours after parasite invasion, the cells were divided into two groups: a blank group treated with DMSO as a control, and a test group treated with 100 μM boscalid. After 24 hours of incubation, the cells were fixed, permeabilized, blocked, and stained. The primary antibody used was rabbit anti-TgGAP45 at a dilution of 1:300, and the secondary antibody used was fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG at a dilution of 1:100. Multiple fields of view were randomly selected under a fluorescence microscope, including the top, bottom, left, right, and center of each well. The number of Toxoplasma tachyzoites within 100 parasite-containing vacuoles was counted. The experiment was repeated three times independently.

[0061] Invasion experiment: HFF cells were collected from 12-well culture plates filled with HFF cells and 1×10 5 RH tachyzoites were divided into two groups. The blank group used DMSO as a control. The experimental group was treated with boscalid at a concentration of 100 μM. After culturing in a 37°C, 5% CO2 incubator for 30 minutes, the culture medium was immediately removed and washed three times with PBS to thoroughly wash away the non-invading tachyzoites. The extracellular tachyzoites could be observed under a microscope to see if they were clean. The culture was continued for 20 hours, and an indirect immunofluorescence assay (IFA) experiment was performed. Place the well under a fluorescence microscope for observation, and 8-10 fields of view were randomly selected from each well, including the top, bottom, left, right, and middle positions of each well. Images of Toxoplasma gondii tachyzoites and host cell nuclei were collected using a fluorescence microscope. The number of parasite-carrying vacuoles and the number of tachyzoite host cells in each picture were counted to express the anti-proliferative effects of boscalid and the control group on intracellular RH tachyzoites, such as Figure 4 The invasion efficiency of each image is the number of parasite-carrying vacuoles per image / the number of host cells per image. The average invasion efficiency of all collected images is the invasion efficiency of the parasite strain. The anti-invasion effect of boscalid and the control group on extracellular RH-type Toxoplasma gondii is shown as follows: Figure 5 shown.

[0062] 2. Experimental results

[0063] After invading host cells, Toxoplasma gondii forms parasitic vacuoles, where tachyzoites multiply by binary fission. Therefore, the number of tachyzoites in the parasitic vacuoles within the same time after invading cells can reflect the parasite's proliferation ability. The statistical results of the proliferation experiment showed that compared with the control group (DMSO group), the parasite invasion and proliferation ability of the group with boscalid was significantly reduced ( Figure 4 、 5 ). This showed that boscalid significantly inhibited the invasion and proliferation of RH Toxoplasma gondii.

[0064] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. Use of boscalid in the preparation of a medicament for preventing or treating Toxoplasma gondii infection.

2. The use of boscalid as claimed in claim 1 in the preparation of a medicament for preventing or treating Toxoplasma gondii infection, characterized in that: The drug for preventing or treating toxoplasmosis is a drug for preventing or treating toxoplasmosis.

3. The use of boscalid as claimed in claim 1 in the preparation of a medicament for preventing or treating Toxoplasma gondii infection, characterized in that: The drug for preventing or treating Toxoplasma infection is a drug that inhibits the proliferation of Toxoplasma or inhibits the ability of Toxoplasma to lyse cells.

4. The use of boscalid as claimed in claim 1 in the preparation of a medicament for preventing or treating Toxoplasma gondii infection, characterized in that: The drug for preventing or treating Toxoplasma infection is a drug that eliminates Toxoplasma gondii in humans or non-human animals.

5. The use of boscalid as claimed in claim 1 in the preparation of a medicament for preventing or treating Toxoplasma gondii infection, characterized in that: By measuring EC 50 When the application amount of boscalid is 40.19 μM, it has a half inhibitory effect on Toxoplasma gondii.

6. Use of boscalid as claimed in claim 1 in the preparation of a medicament for preventing or treating Toxoplasma gondii infection, characterized in that: When the dosage of boscalid is 10000 μM, it has no cytotoxicity to Vero cells.

7. A composition for preventing or treating Toxoplasma gondii infection, characterized in that: The active ingredient of the composition includes boscalid.

8. The composition for preventing or treating Toxoplasma gondii infection according to claim 7, wherein The composition further comprises one or more of a diluent, a wetting agent, a binder, a disintegrant, a lubricant, a color and flavor regulator, a solvent, a solubilizer, a cosolvent, an emulsifier, an antioxidant, a metal complexing agent, a preservative, a pH regulator, a surfactant, an excipient, a filler and a synergist.

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