Application of phenothiazine in preparation of medicine for treating toxoplasmosis

Abstract

The invention provides application of phenothiazine in preparation of a medicine for treating toxoplasmosis, and particularly relates to application of phenothiazine 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 phenothiazine. The inventor finds and verifies that phenothiazine 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 serve as cells of an experimental model, and EC50 determination shows that phenothiazine has a half of the inhibitory effect on the toxoplasma gondii when the application amount is 29.00 mu M. Meanwhile, when the application amount of phenothiazine is 5000 [mu] M, the phenothiazine has no cytotoxicity to Vero cells. Compared with existing drugs for treating toxoplasma gondii infection, the effective inhibition concentration of phenothiazine provided by the invention is far smaller than the cytotoxicity of phenothiazine, and phenothiazine used for preparing drugs for preventing or treating toxoplasma gondii infection has the remarkable advantages of high efficiency, low toxicity and safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of parasitic disease prevention and treatment, and relates to the application of phenothiazines in preventing and treating Toxoplasma gondii infection, and specifically relates to the application of phenothiazines in preparing drugs 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 phenothiazines in the preparation of drugs for preventing or treating Toxoplasma infection, which specifically include:

[0007] One of the purposes of the present invention is to provide the use of phenothiazine in the preparation of a drug for preventing or treating Toxoplasma gondii infection. The CAS number of phenothiazine is 92-84-2, and its molecular formula is C 12 H9NS, chemical structure:

[0008]

[0009] Phenothiazine is an organic compound with a tricyclic structure. It serves as both the core structure of antipsychotic drugs and an important chemical synthesis intermediate. Its chemical structure consists of two benzene rings connected by a sulfur and nitrogen atom to form a tricyclic ring. This core structure determines its lipid solubility and receptor binding ability. In medicine, phenothiazine is suitable for the symptomatic treatment of acute and chronic schizophrenia, mania, reactive psychosis, and other severe mental illnesses, effectively controlling symptoms such as excitement, aggression, hallucinations, delusions, and impaired thought association. In industry, phenothiazine is an intermediate in the manufacture of methylene blue dye and an excellent polymerization inhibitor in the production of olefinic monomers such as acrylates, methacrylates, and vinyl acetate. It is also used as an additive for synthetic materials, such as a polymerization inhibitor in the production of vinylon, and is also a raw material for rubber antioxidants. However, there are no reports of phenothiazine's use against Toxoplasma gondii.

[0010] In a preferred embodiment, the drug for preventing or treating Toxoplasma gondii infection is a drug for preventing or treating toxoplasmosis.

[0011] In a preferred embodiment, 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.

[0012] 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.

[0013] In a preferred embodiment, by measuring EC 50 When the phenothiazine dosage is 29.00 μM, it has half the inhibitory effect on Toxoplasma gondii.

[0014] In a preferred embodiment, the phenothiazine has no cytotoxicity to Vero cells when administered at a dose of 5000 μM.

[0015] 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 phenothiazine.

[0016] 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.

[0017] 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.

[0018] In the present invention, the dosage forms of the composition include, but are not limited to, powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, and other oral preparations, external preparations, suppositories, and sterile injectable solutions. It will be appreciated that the phenothiazine-containing composition of the present invention can be administered in a variety of ways, depending on the different excipients and dosage forms.

[0019] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0020] 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 inventors, through extensive research, have discovered that phenothiazines have 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 phenothiazines.

[0021] 2. The present inventors have verified through a large number of experiments that phenothiazines have significant effects 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 phenothiazine had a 50% inhibitory effect on Toxoplasma gondii at 29.00 μM. Therefore, a small amount of application can achieve a highly effective anti-Toxoplasma infection effect.

[0022] 3. Furthermore, experiments have confirmed that phenothiazines, as anti-Toxoplasma drugs, have the advantage of low toxicity. Specifically, the cytotoxicity of phenothiazines to green monkey kidney (Vero) cells was tested using the CCK8 assay. The results showed that phenothiazines were not cytotoxic to Vero cells at a maximum dose of 5000 μM. Therefore, compared to existing drugs for treating Toxoplasma infection, which suffer from significant side effects, the phenothiazines provided in the present invention have an effective inhibitory concentration far below their cytotoxicity, and thus possess the significant advantages of being highly effective, low-toxic, and safe for use in the preparation of drugs for the prevention or treatment of Toxoplasma infection.

[0023] 4. The present invention has found through a large number of experimental explorations that the anti-Toxoplasma effect of phenothiazine is mainly through inhibiting its intracellular proliferation, and the effect of phenothiazine on Toxoplasma is dose-dependent, indicating that the higher the concentration, the better the anti-insect effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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:

[0025] Figure 1 This is a graph showing the results of a toxicity test of phenothiazine on Vero cells in Example 1 of the present invention;

[0026] Figure 2 This is a graph showing the inhibitory effect of phenothiazines on Toxoplasma gondii in Example 2 of the present invention;

[0027] Figure 3 This is a plaque assay diagram of phenothiazine and a control group in Example 3 of the present invention;

[0028] Figure 4This is a statistical graph showing the anti-proliferative effects of phenothiazine and a control group on intracellular RH tachyzoites in Example 4 of the present invention;

[0029] Figure 5 This is a graph showing the anti-invasion effect of phenothiazine and the control group on extracellular RH-type Toxoplasma gondii in Example 4 of the present invention. DETAILED DESCRIPTION

[0030] 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.

[0031] The present invention provides the use of phenothiazines in the preparation of drugs 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, large toxic side effects, and poor effect in preventing and treating toxoplasmosis infection.

[0032] The present invention uses phenothiazines, which are conventionally used as intermediates for pharmaceutical or chemical products, to eliminate Toxoplasma gondii parasites in humans or non-human animals, thereby avoiding the problem of drug resistance caused by long-term use of traditional anti-insect drugs. In addition, because the effective inhibitory concentration of phenothiazines on Toxoplasma gondii is far lower than its cytotoxicity, a highly effective, low-toxic and safe drug for anti-Toxoplasma infection is provided.

[0033] The technical solution of this application is described in detail below through specific embodiments:

[0034] Unless otherwise specified, the techniques used in the present invention are conventional techniques 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. Phenothiazine used in the present invention was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., and Vero cells and HFF cells used in the present invention were purchased from the ATCC cell bank.

[0035] Example 1

[0036] Study on the toxicity of phenothiazines to Vero cells

[0037] 1. Experimental process

[0038] Cytotoxicity assay: The CCK-8 assay was used to determine the toxicity of phenothiazines to Vero cells. A 96-well cell culture plate was inoculated with a suspension of African green monkey kidney (Vero) cells, approximately 100 μL (5,000 Vero cells) per well. The culture plate was placed in an incubator for a pre-culture period (37°C, 5% CO2) to allow the cells to adhere for approximately four hours. Eight concentration gradients ranging from 40 μM to 5,000 μM were set up, and Vero cells were treated with phenothiazines at different concentrations 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 using the following formula. The independent experiment was repeated three times.

[0039] Cell viability (%) = (OD 实验组 -OD 空白组 ) / (OD 对照组 -OD 空白组)×100％

[0040] 2. Experimental results

[0041] like Figure 1 The absorbance values of African green monkey kidney cells treated with different concentrations of phenothiazine at 450nm were statistically analyzed. The results showed that there was no significant difference in the absorbance between the phenothiazine treatment groups and the DMSO control group, indicating that phenothiazine has low toxicity and safety to cells.

[0042] Example 2

[0043] Inhibitory Effects of Phenothiazines on Toxoplasma Gondii

[0044] 1. Experimental process

[0045] 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 phenothiazine from 0 to 1000 μM were added. The culture medium in each well was controlled at 150-300 μL, and the independent experiment was repeated 3 times. After culturing for 24 hours, 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 in a chemiluminescence instrument to calculate the survival rate of Luciferase.

[0046] Inhibition rate % = (RLU DMSO -RLU 处理组 ) / RLU DMSO ×100

[0047] 2. Experimental results

[0048] like Figure 2 The results showed that the half maximum effective concentration (EC50) of phenothiazine against Toxoplasma gondii 50 ) is 29.00 μM. Furthermore, the inhibitory effect on Toxoplasma gondii is dose-dependent, with higher concentrations increasing the inhibitory effect. This suggests that phenothiazines have a good inhibitory effect on Toxoplasma gondii.

[0049] Example 3

[0050] Effects of phenothiazines on the inhibition of intracellular plaque formation of Toxoplasma gondii

[0051] 1. Experimental process

[0052] 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 90 μM phenothiazine to each well and incubated for 7 days (the culture medium in each well was controlled at 2-3 mL). The cells 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.

[0053] 2. Experimental results

[0054] 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 phenothiazine group, indicating that phenothiazine significantly inhibited the growth of Toxoplasma gondii.

[0055] Example 4

[0056] Antiproliferative Effects of Phenothiazines on Intracellular Toxoplasma Gondii Type I Strain (RH)

[0057] 1. Experimental process

[0058] Proliferation assay: Collect 1×10 5 RH tachyzoites were inoculated into a 12-well plate filled with a monolayer of HFF cells (the ratio of the number of Toxoplasma gondii to the number of host cells was 1:10). After 4 hours of invasion, the cells were divided into two groups. The blank group used DMSO as a control, and the experimental group was treated with phenothiazine at a concentration of 90 μM (3 times the EC 50After 24 hours of incubation, cells were fixed, permeabilized, blocked, and stained. The primary antibody used was rabbit-derived Toxoplasma surface protein GAP45, diluted 1:300, and the secondary antibody used was fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit fluorescent secondary antibody, diluted 1:100. Observe under a fluorescence microscope. Multiple fields of view were randomly selected, 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.

[0059] 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, and the experimental group was treated with phenothiazine at a concentration of 90 μM (3 times EC 50 ). After culturing in a 37°C, 5% CO2 incubator for 30 minutes, the culture medium was immediately removed and the cells were washed three times with PBS to thoroughly wash away the non-invading tachyzoites. The extracellular tachyzoites can be observed under a microscope to see if they have been cleaned. Continue culturing for 20 hours and perform an indirect immunofluorescence assay (IFA) experiment. Place the cells under a fluorescence microscope for observation, and randomly select 8-10 fields of view from each well, including the top, bottom, left, right, and middle positions of each well. Use a fluorescence microscope to capture images of Toxoplasma tachyzoites and host cell nuclei. Count the number of parasite-carrying vacuoles and the number of tachyzoite host cells in each picture, and express the anti-proliferative effects of phenothiazine 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 phenothiazine and the control group on extracellular RH-type Toxoplasma is shown as follows: Figure 5 shown.

[0060] 2. Experimental results

[0061] 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 phenothiazine was significantly reduced ( Figure 4 、 5 ). This indicates that phenothiazines significantly inhibit the invasion and proliferation of RH Toxoplasma gondii.

[0062] 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 phenothiazines in the preparation of drugs for preventing or treating Toxoplasma gondii infection.

2. Use of the phenothiazine according to 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. Use of the phenothiazine according to claim 1 in preparing 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. Use of the phenothiazine according to claim 1 in preparing 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. Use of the phenothiazine according to claim 1 in preparing a medicament for preventing or treating Toxoplasma gondii infection, characterized in that: By measuring EC 50 When the phenothiazine dosage is 29.00 μM, it has half the inhibitory effect on Toxoplasma gondii.

6. Use of the phenothiazine according to claim 1 in preparing a medicament for preventing or treating Toxoplasma gondii infection, characterized in that: When the phenothiazine is administered at a dose of 5000 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 a phenothiazine.

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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