Application of dihydrochloride in preparation of medicine for preventing and treating toxoplasmosis

Abstract

The invention relates to the technical field of biology, in particular to application of dihydrochloride in preparation of medicine for preventing and treating toxoplasmosis, through exploration, it is found that the dihydrochloride has a safe and effective inhibition effect on toxoplasma gondii and also has a remarkable inhibition effect on invasion of the toxoplasma gondii, the splitting cycle of the toxoplasma gondii can be destroyed, and the effect of preventing and treating toxoplasmosis is achieved. The acute toxoplasmosis is effectively treated, and a new thought is provided for research and development of a new safe and effective medicine for treating the acute toxoplasmosis.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the application of dihydrochloride in preparing medicines for preventing and treating toxoplasmosis. Background Art

[0002] Toxoplasmosis is a global zoonotic parasitic disease caused by Toxoplasma gondii, which is particularly prevalent in warm and humid areas. The disease is mainly transmitted through ingestion of food or water contaminated by cat feces containing Toxoplasma oocysts, and can also be transmitted through mother-to-child vertical transmission or organ transplantation. Toxoplasmosis can cause a variety of symptoms in the human body, ranging from mild flu-like symptoms to severe central nervous system damage, and even life-threatening acute infection, especially in immunocompromised people. Although existing treatments such as sulfonamides and pyrimethamine can control the disease to a certain extent, they have obvious side effects and limited therapeutic effects on acute toxoplasmosis, especially the lack of effective inhibitors against the invasion and lytic cycle of Toxoplasma. Therefore, finding and developing new safe and effective anti-Toxoplasma drugs has become a hot topic and urgent need for current research.

[0003] Dihydrochloride (SCH79797), its CAS number is: 245520-69-8, has a wide range of application potential in the field of medicine. It can exert biological activity through different mechanisms. In recent years, more and more studies have shown that dihydrochloride has antibacterial, antiviral and even antiparasitic activity, and is relatively safe with little side effects. However, regarding the specific application of dihydrochloride in the treatment of toxoplasmosis and its mechanism of action, current research is still insufficient, especially lacking in its in-depth exploration in suppressing toxoplasma invasion, destroying toxoplasma lysis cycle and treating acute toxoplasmosis.

[0004] Therefore, exploring the application of dihydrochloride in the preparation of drugs for the prevention and treatment of toxoplasmosis, especially its potential in safely and effectively inhibiting the invasion of Toxoplasma gondii, destroying the Toxoplasma lysis cycle and treating acute toxoplasmosis, not only provides new ideas for the development of new anti-Toxoplasma drugs, but also provides new possibilities for solving the current problems in the treatment of toxoplasmosis. Summary of the invention

[0005] The purpose of the present invention is to provide an application of a dihydrochloride in the preparation of a drug for preventing and treating toxoplasmosis, which has a safe and effective inhibitory effect on Toxoplasma gondii and also has a significant inhibitory effect on the invasion of Toxoplasma gondii.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] The invention provides an application of a dihydrochloride salt in preparing a medicine for preventing and treating toxoplasmosis.

[0008] Furthermore, the toxoplasmosis is acute toxoplasmosis.

[0009] Furthermore, the dihydrochloride prevents and treats toxoplasmosis by inhibiting Toxoplasma gondii and its invasion and destroying the lytic cycle of Toxoplasma gondii.

[0010] Furthermore, the dosage forms of the drug include tablets, capsules, oral preparations and injections.

[0011] Furthermore, the tablet comprises a coated tablet.

[0012] Furthermore, the oral preparation includes oral liquid, oral granules and oral powder.

[0013] Furthermore, the injection includes freeze-dried powder injection and injection emulsion.

[0014] Beneficial effects:

[0015] The present invention has found through exploration that the dihydrochloride has a safe and effective inhibitory effect on Toxoplasma gondii, also has a significant inhibitory effect on the invasion of Toxoplasma gondii, and can destroy the lysis cycle of Toxoplasma gondii, effectively treating acute toxoplasmosis, and provides a new idea for the development of new safe and effective drugs for the treatment of acute toxoplasmosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 The results of the cytotoxicity of SCH79797 to HFF cells and the inhibition of Toxoplasma gondii are shown in Figure 1, where A is the cytotoxicity of SCH79797 to HFF cells, and B is the inhibition of SCH79797 to Toxoplasma gondii. SI = CC 50 / EC 50 , *** indicates P < 0.001;

[0018] Figure 2 This is the result of the anti-invasion effect of SCH79797 on Toxoplasma gondii in vitro, where **** means P<0.0001;

[0019] Figure 3 This is the result of the plaque test, where the scale is 2 mm;

[0020] Figure 4The results of the effects of SCH79797 on the ultrastructure of Toxoplasma gondii are shown in Figure 1, where A is the effect of SCH79797 and the control group on the endogenous stage of Toxoplasma gondii, and B is the effect of SCH79797 and the control group on the intrazonal vesicle of Toxoplasma gondii, with a scale of 1 μm;

[0021] Figure 5 This is a graph showing the therapeutic effect of SCH79797 on mice with acute toxoplasmosis, where A is the weight change of mice with acute toxoplasmosis, B is the survival status of mice with acute toxoplasmosis, and SUL is sulfadiazine. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] The test methods used in the following implementations, unless otherwise specified, are all commonly used experimental methods in this field;

[0028] Unless otherwise specified, the experimental materials used in the following examples are all commonly used experimental materials in this field.

[0029] Example 1 Verification of the therapeutic effect of SCH79797 on toxoplasmosis

[0030] 1. Verification method

[0031] 1.1 Cytotoxicity test of SCH79797

[0032] The toxicity of SCH79797 to HFF cells was determined by CCK-8 assay. 4 / mL, inoculated in a 96-well plate, cultured for 12 hours, and then added 32, 16, 8, 4, 2, 1, 0.5 and 0.25μM SCH79797. The control group is the wells with cells and culture medium, and the wells containing only culture medium are blank controls to correct the background. After 72 hours of culture, CCK-8 reagent was added, and the absorbance value of each well was measured at a wavelength of 450nm on a microplate reader after 1 hour.

[0033] 1.2 Inhibition test of SCH79797 on Toxoplasma gondii

[0034] RH-2F tachyzoites expressing β-galactosidase were used for growth inhibition assays. Fresh RH-2F tachyzoites were harvested from HFF cells and the concentration of tachyzoites was adjusted to 2 × 10 3 / mL. 500, 250, 62.5 and 31.25nM SCH79797 were prepared with this, and 0.1% DMSO was used as the control group. SCH79797 and tachyzoites were added to the monolayer of HF F cells in a 96-well plate, and 10μL of 10% Triton X-100 and 1mM CPRG were added to each well after incubation for 72h. Incubate in the incubator for another 12h, and then measure the absorbance at 570nm.

[0035] 1.3 SCH79797 Invasion Test of Toxoplasma gondii

[0036] Use 4×10 5 500, 250, 62.5 and 31.25 nM SCH79797 and 0.1% DMSO were prepared from RH-2F tachyzoites with a concentration of 100 / mL and added to a 96-well plate containing HFF cells. After 2 hours, the uninvaded tachyzoites were washed with PBS and the culture medium was added again. Finally, 10 μL of 10% Triton X-100 and 1 mM CPRG were added. Incubate in the incubator for another 12 hours, and then measure the absorbance at 570 nm. The invasion rate in the 0.1% DMSO control group was calculated as 100%, and the invasion rates in other groups were calculated accordingly.

[0037] 1.4 Plaque test

[0038] 300 RH tachyzoites were added to a 6-well plate containing HFF cells and left to stand for 2 hours. Then 0.5 μM SCH79797 was added, and 0.1% DMSO was used as a control. After 7 days, the cells were fixed with cold methanol for 15 minutes at room temperature, then washed with PBS, stained with 1% crystal violet for 15 minutes, washed with PBS, and photographed.

[0039] 1.5 Transmission electron microscopy observation

[0040] RH tachyzoites were added to Vero cells in 75T culture flasks at a ratio of 2:1. 0.5μM SCH79797 was added 12 hours later, and 0.1% DMSO was used as the control group. After incubation for 24 hours, the liquid in the cell culture flask was discarded, and trypsin was added after washing twice with PBS for digestion. After terminating the digestion, centrifugation was performed, and PBS was washed twice. The cells and parasites were precipitated and fixed with 2.5% glutaraldehyde at room temperature for 30 minutes, and then transferred to a 4°C refrigerator for fixation overnight. Then, 1% agarose was used for pre-embedding, and 1% osmium acid was used for fixation at room temperature in the dark for 2 hours. After rinsing with PBS, dehydration, penetration embedding and polymerization were performed. The resin block was cut into 60-80nm ultrathin sections in an ultrathin slicer, and the sections were stained in 2% uranyl acetate and 2.6% lead citrate solutions for 8 minutes each. After rinsing, they were dried at room temperature overnight. Finally, the sections were observed and photographed under a transmission electron microscope.

[0041] 1.6 Treatment of acute toxoplasmosis in mice

[0042] Thirty-six female BALB / c mice were randomly divided into three groups. Each mouse was intraperitoneally injected with 1000 RH tachyzoites, and drug administration began 4 hours later. The experiment was divided into a SCH79797 treatment group (5 mg / kg / d), a positive drug sulfadiazine control group (100 mg / kg / d), and a negative control group of normal saline. SCH79797 was dissolved in 0.5% sodium carboxymethyl cellulose, and sulfadiazine was dissolved in normal saline. The drug was administered by gavage every day for 7 consecutive days. The weight changes of each group in the first eight days and the survival of the sick mice within 60 days were recorded.

[0043] 2. Conclusion

[0044] 2.1 Cytotoxicity test results of SCH79797

[0045] The results are as follows Figure 1 As shown in Figure A, it can be seen that the cytotoxicity of SCH79797 to HFF is dose-dependent. The higher the concentration of SCH79797, the lower the survival rate of HFF cells. When the concentration of SCH79797 is 0.5 μM, the cell survival rate of HFF is 103.05%, indicating that SCH79797 is not toxic to HFF cells at this concentration.

[0046] 2.2 Results of the Inhibitory Test of SCH79797 on Toxoplasma gondii

[0047] The results are as follows Figure 1 As shown in Figure B, it can be seen that the inhibitory effect of SCH79797 on Toxoplasma tachyzoites is also dose-dependent. When the concentration of SCH79797 is 0.5 μM, the survival rate of Toxoplasma RH-2F is only 9.91%. According to the formula SI=CC50 / EC50, the selectivity index of SCH79797 is 139.71, which is much greater than the standard 1, indicating that SCH79797 has a safe and effective inhibitory effect on Toxoplasma.

[0048] 2.3 Results of SCH79797 test on Toxoplasma gondii invasion

[0049] The results are as follows Figure 2 As shown in the figure, it can be seen that SCH79797 has a significant inhibitory effect on the invasion of Toxoplasma gondii. Consistent with the inhibition test, SCH79797 is also dose-dependent on the invasion of Toxoplasma gondii. The invasion rate of Toxoplasma tachyzoites at a concentration of 0.5 μM is the lowest, at 13.29%.

[0050] 2.4 Plaque test results

[0051] The results are as follows Figure 3 As shown in the figure, it can be seen that SCH79797 can significantly disrupt the lytic cycle of Toxoplasma gondii. In the control group, Toxoplasma gondii formed many plaques in the monolayer of HFF cells, while the SCH79797 group had a normal monolayer of HFF cells.

[0052] 2.5 Transmission electron microscopy observation results

[0053] In the control group, Toxoplasma had normal morphology and was observed to be in the endogenous stage ( Figure 4 In the middle (A) of Toxoplasma gondii, the intrazonal vesicles were also observed ( Figure 4 In contrast, in the SCH79797 group, many Toxoplasma gondii with abnormal morphology were observed, with deformed nuclei and very typical autophagic structures (red arrows).

[0054] 2.6 Results of the acute toxoplasmosis treatment experiment in mice

[0055] The mice in the SCH79797 and negative control groups basically began to become ill on the 6th day, with messy hair, listlessness, and weight loss. The mice in the negative control group began to die on the eighth day, and all the mice died on the tenth day. The mice in the SCH79797 group began to die from the eighth day, and 3 mice were still alive at 60 days. In the positive control drug sulfadiazine (SUL) group, the mice died between the ninth and thirtieth days, and 3 mice were alive at 60 days. The survival rate of the mice in the SCH7979 group was comparable to that of the positive control drug sulfadiazine, both at 40%. The specific results are as follows: Figure 5 As shown, it was shown that SCH79797 had a certain therapeutic effect on mice with acute toxoplasmosis.

[0056] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of dihydrochloride in the preparation of drugs for preventing and treating toxoplasmosis.

2. The use according to claim 1, characterized in that The toxoplasmosis is acute toxoplasmosis.

3. The use according to claim 1, characterized in that The dihydrochloride prevents and treats toxoplasmosis by inhibiting Toxoplasma gondii and its invasion and destroying the lytic cycle of Toxoplasma gondii.

4. The use according to claim 1, characterized in that The dosage forms of the drug include tablets, capsules, oral preparations and injections.

5. The use according to claim 4, characterized in that: The tablets include coated tablets.

6. The use according to claim 4, characterized in that: The oral preparation includes oral liquid preparation, oral granule preparation and oral powder preparation.

7. The use according to claim 4, characterized in that: The injection includes freeze-dried powder injection and injection emulsion.

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