Application of compound triclosan in preparation of medicine for treating toxoplasmosis

Abstract

The invention discloses application of a compound triclosan in preparation of a medicine for treating toxoplasmosis. The medicine is used for inhibiting toxoplasmosis proliferation. The medicine is used for relieving pathological injuries of one or more organs caused by toxoplasma gondii infection, and the organs comprise the heart, the liver, the spleen, the kidney and the brain. The medicine is used for protecting brain tissues or penetrating a blood brain barrier to treat toxoplasma gondii infection of a central nervous system; the medicine comprises a therapeutically effective amount of triclosan and a pharmaceutically acceptable carrier or auxiliary material, the dosage form of the medicine is an oral preparation; the application has the beneficial effects that the broad-spectrum antibacterial agent triclosan is proved to have a new application of remarkably treating toxoplasmosis for the first time, in-vivo and in-vitro experiments prove that triclosan can effectively inhibit the proliferation of toxoplasmosis, the effect is dose-dependent, and in an acute infection mouse model, triclosan can remarkably improve the survival rate of mice and reduce the ascites insect-bearing quantity, so that the effect of treating toxoplasmosis is achieved. The pathological injury of multiple organs such as heart, liver, spleen and kidney can be relieved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically the application of the compound triclosan in the preparation of drugs for treating toxoplasmosis. Background Technology

[0002] Toxoplasma gondii is a strictly intracellular opportunistic pathogenic protozoan that widely parasitizes nucleated cells in humans and animals. It can infect almost all warm-blooded animals, including humans, such as pigs, cattle, sheep, horses, and camels. Felines are its definitive hosts. Global serological epidemiological surveys show that approximately one-third of the population is antibody-positive, with immunocompromised individuals and pregnant women at higher risk. If a pregnant woman is infected with Toxoplasma gondii in early pregnancy, it can lead to miscarriage, stillbirth, or birth defects. Furthermore, Toxoplasma gondii exhibits significant neurotropic and retinal-tropic properties, often causing serious complications such as encephalitis and retinochoroiditis, which can be fatal in severe cases, posing a significant threat to human health and livestock farming. In clinical treatment, the combination of sulfadiazine and pyrimethamine is currently the first-line therapy; however, this method has poor cyst penetration and significant side effects on organs such as the liver and kidneys, including bone marrow suppression and allergic reactions. It is also ineffective against cysts in the chronic infection stage. Existing literature indicates that Toxoplasma gondii has developed resistance to sulfonamides, and currently used treatments are insufficient to completely eradicate the parasites, leading to a high relapse rate after discontinuation of medication. Therefore, developing novel, safe, and highly effective anti-Toxoplasma gondii drugs, especially those that overcome the aforementioned shortcomings of existing therapies, has become an urgent need in clinical treatment and public health.

[0003] Triclosan is a broad-spectrum antibacterial agent that has been widely added to personal care products such as antibacterial soaps, toothpastes, mouthwashes, and shower gels. However, there are no reports of its use in treating toxoplasmosis. Therefore, we propose the application of triclosan in the preparation of drugs for treating toxoplasmosis. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing anti-toxoplasmosis drugs (such as sulfadiazine) and provide a new pharmaceutical use for the compound triclosan, specifically the application of the compound triclosan in the preparation of drugs for treating toxoplasmosis, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the use of the compound triclosan in the preparation of a medicament for treating toxoplasmosis.

[0006] Preferably, the drug is used to inhibit the proliferation of Toxoplasma gondii.

[0007] Preferably, the drug is used to alleviate pathological damage to one or more organs caused by Toxoplasma gondii infection, including the heart, liver, spleen, kidneys, and brain.

[0008] Preferably, the drug is used to protect brain tissue or to penetrate the blood-brain barrier to treat toxoplasmosis infection of the central nervous system.

[0009] Preferably, the drug comprises a therapeutically effective amount of triclosan and a pharmaceutically acceptable carrier or excipient.

[0010] Preferably, the dosage form of the drug is an oral formulation.

[0011] Preferably, when the drug is administered to an individual, the daily dose of triclosan is 50 mg / kg to 200 mg / kg body weight.

[0012] Preferably, the drug is used in combination with at least one other anti-Toxoplasma gondii drug.

[0013] Compared with the prior art, the beneficial effects of the present invention are:

[0014] 1. This invention is the first to demonstrate that the broad-spectrum antibacterial agent triclosan has a significant new application in the treatment of toxoplasmosis. In vitro and in vivo experiments have shown that triclosan effectively inhibits the proliferation of Toxoplasma gondii in a dose-dependent manner. In an acute infection mouse model, triclosan (especially the high-dose group of 200 mg / kg) significantly improved mouse survival rate, reduced the amount of Toxoplasma gondii in ascites, and alleviated pathological damage to multiple organs, including the heart, liver, spleen, and kidneys. Most importantly, triclosan exhibits a protective effect on brain tissue, suggesting its potential to penetrate the blood-brain barrier, providing a possible treatment for brain complications caused by Toxoplasma gondii.

[0015] 2. Compared with the traditional first-line drug sulfadiazine sodium, this invention demonstrates that triclosan exhibits better therapeutic efficacy and higher safety in improving the survival rate of infected mice and reducing damage to organs such as the liver and kidneys. This invention provides a novel, potent, and safe drug candidate to address the problems of poor blood-brain barrier penetration, significant toxic side effects, and increasingly serious drug resistance associated with existing therapies. This "drug repurposing" strategy not only shortens the drug development cycle but also brings new hope to the clinical treatment of toxoplasmosis. Attached Figure Description

[0016] Figure 1 For the cytotoxicity test of this invention;

[0017] Figure 2 The CCK-8 method of this invention is used to detect the in vitro anti-Toxoplasma gondii effect of triclosan;

[0018] Figure 3 The plaque assay method of this invention is used to detect the in vitro anti-Toxoplasma gondii effect of triclosan;

[0019] Figure 4This is the survival time curve of mice after triclosan treatment according to the present invention;

[0020] Figure 5 The amount of *Helicobacter pylori* in the ascites of mice after triclosan treatment according to the present invention;

[0021] Figure 6 The present invention relates to PCR detection of organ infection in mice;

[0022] Figure 7 Histopathological observation of mice in each group according to the present invention. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] This invention provides a technical solution: the application of the compound triclosan in the preparation of drugs for treating toxoplasmosis.

[0025] The drug is used to inhibit the proliferation of Toxoplasma gondii.

[0026] The drug is used to alleviate pathological damage to one or more organs caused by Toxoplasma gondii infection, including the heart, liver, spleen, kidneys, and brain.

[0027] The drug is used to protect brain tissue or to penetrate the blood-brain barrier to treat toxoplasmosis infection of the central nervous system.

[0028] The drug contains a therapeutically effective amount of triclosan and a pharmaceutically acceptable carrier or excipient.

[0029] The drug is an oral formulation.

[0030] When administered to an individual, the daily dose of triclosan is 50 mg / kg to 200 mg / kg body weight.

[0031] The drug is used in combination with at least one other anti-toxoplasmosis drug.

[0032] Please see Figure 1-3 Example 1: Evaluation of the in vitro anti-Toxoplasma gondii activity and cytotoxicity of triclosan.

[0033] ① Cell and insect strain culture:

[0034] Vero cells were resuscitated and cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37°C with 5% CO2. When the cells reached approximately 90% confluence with the bottom of the flask, they were passaged using trypsin digestion. The Toxoplasma gondii RH strain (F11 generation) was resuscitated from liquid nitrogen, resuspended in DMEM medium containing 1% FBS, and then inoculated into Vero cells that had formed a dense monolayer for in vitro culture. After a large amount of tachyzoites were released, they were scraped off with a cell scraper and repeatedly aspirated with a 27G needle to release the parasites. The cells were then collected by sequential centrifugation at 200g for 5 minutes, filtration through a 5μm filter, and centrifugation at 800g for 10 minutes. After three stable passages, the tachyzoites were counted for subsequent experiments.

[0035] ②Cytotoxicity test:

[0036] The toxicity of triclosan to Vero cells was determined using the CCK-8 assay. Triclosan was dissolved in DMSO to prepare a stock solution of 1 g / mL, which was then diluted with DMEM medium containing 1% FBS to a series of concentration gradients (50, 100, 200, 400, 500, 600, 800, 1000 μg / mL) for the experiment. Vero cells were incubated at 1 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of [number] cells / mL in 96-well plates and cultured for 12 hours to form a monolayer. The medium was then replaced with culture medium containing different concentrations of triclosan, and a control group (no drug added) and a blank group (containing only culture medium) were established. After culturing for another 24 hours, CCK-8 reagent was added to each well, and the cells were incubated for 2 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader. The cell proliferation rate was calculated using the formula: [Cell proliferation rate (%) = (OD experimental group - OD blank group) / (OD control group - OD blank group) × 100%]. The results showed that the maximum safe concentration of triclosan for Vero cells (cell proliferation rate > 90%) was 600 μg / mL.

[0037] ③ In vitro anti-Toxoplasma gondii test:

[0038] Vero cells were seeded into 96-well plates and cultured into a monolayer, with a graft density of 5 × 10⁶ cells / well. 3A suspension of *Toxoplasma gondii* tachyzoites per mL was incubated for 8 hours to induce parasite invasion into cells. Uninvaded parasites were removed by washing twice with PBS. Fresh culture medium containing different concentrations of triclosan was then added to each well at concentrations below the maximum safe concentration (600 μg / mL). A control group without the drug was also included. After culturing at 37°C and 5% CO2 for 48 hours, the supernatant was discarded, and 100 μL of fresh culture medium and 10 μL of LCK-8 solution were added to each well. After incubation for 2 hours, the OD value at 450 nm was measured. The inhibition rate against *Toxoplasma gondii* was calculated using the formula: [Relative inhibition rate (%) = (OD control group - OD drug group) / OD control group × 100%]. The results showed that triclosan at 600 μg / mL inhibited *Toxoplasma gondii* intracellularly by up to 79.6%, and the inhibitory effect was dose-dependent, significantly superior to the control drug sulfadiazine sodium.

[0039] ④ Plaque test:

[0040] Vero cells were loaded at 2 × 10 5 Inoculated at a density of *Toxoplasma gondii* tachyzoites per mL into 6-well plates and cultured to a monolayer, then inoculated with an equal amount of *Toxoplasma gondii* tachyzoites and incubated for 8 hours. After discarding the culture medium and washing with PBS, media containing 200 μg / mL triclosan or 200 μg / mL sulfadiazine sodium (positive control) were added, with a blank control included. At 24, 48, and 72 hours after drug addition, the culture medium was discarded, the plates were washed with PBS, stained with Giemsa stain, and observed and photographed under an inverted microscope (×40). The results showed that, at the same concentration and after 72 hours of treatment, the number of *Toxoplasma gondii* larvae in the triclosan-treated group was significantly less than that in the sulfadiazine sodium group and the blank control, further confirming the excellent direct in vitro antiparasitic effect of triclosan.

[0041] Please see Figure 4-7 Example 2: Evaluation of the in vivo anti-Toxoplasma gondii effect of triclosan.

[0042] ① Animal model construction and drug administration:

[0043] The experimental mice were randomly divided into 5 groups of 10 mice each:

[0044] Group 1: High-dose triclosan group (200 mg / kg);

[0045] Group 2: Triclosan medium-dose group (100mg / kg);

[0046] Group 3: Low-dose triclosan group (50 mg / kg);

[0047] Positive control group (sulfadiazine sodium, 100 mg / kg);

[0048] Blank control group (equal volume of corn oil solvent);

[0049] All drugs used corn oil as a solvent. Mice in each group were intraperitoneally inoculated with 1×10⁻⁶ mmol / L. 4 Toxoplasma gondii RH tachyzoites per mL were administered via gavage starting 4 hours after infection, once daily for 5 consecutive days.

[0050] ② Survival rate and ascites worm count detection:

[0051] During the 7-day observation period, the survival status of mice was recorded daily. On days 1, 3, 5, and 7 post-infection, one mouse from each group was randomly selected, and ascites fluid was collected. The number of Toxoplasma gondii in the ascites fluid was counted under a microscope using a hemocytometer. The results are shown in Table 1.

[0052] Table 1. In vivo efficacy of the drug against Toxoplasma gondii

[0053]

[0054] The results showed that the high-dose triclosan group (200 mg / kg) had the highest survival rate (40%), which was significantly better than the positive control group (20%) and the blank control group (10%). In terms of the amount of cephalosporins in the ascites fluid, the high-dose triclosan group showed a significant worm-inhibiting effect on day 7, and the worm load was comparable to that of the sodium sulfadiazine group, indicating that it has strong anti-worm activity in vivo.

[0055] ③ PCR detection of worm load in organ tissues:

[0056] Seven days after treatment, heart, liver, spleen, lung, kidney, and brain tissues were collected from mice in each group, and genomic DNA was extracted. PCR amplification was performed using Toxoplasma gondii B1 gene-specific primers. The reaction volume was 25 μL, containing 12.5 μL Taq MasterMix, 1 μL each of forward and reverse primers, 1 μL template DNA, and 9.5 μL ddH2O. The PCR program was: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 10 seconds, 60℃ annealing for 10 seconds, 72℃ extension for 20 seconds, for a total of 35 cycles; and a final extension at 72℃ for 5 minutes. The amplified products were analyzed by 2% agarose gel electrophoresis. The results showed that Toxoplasma gondii DNA was not detected in the heart and brain tissues of mice in the high-dose triclosan group. The band brightness detected in the liver, lung, and kidney tissues was also weaker than that in the control group, indicating that triclosan can effectively inhibit or reduce the parasitic infection of Toxoplasma gondii in vital organs, especially showing good protective potential in the heart and brain tissues.

[0057] ④ Organ pathological examination:

[0058] On day 7 of infection, heart, liver, spleen, lung, kidney and brain tissues of mice in each group were collected, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned and stained with HE, and then the pathological changes were observed under an optical microscope (×200).

[0059] Liver: Microgranulomas or granulomatous lesions were observed in the blank control group; multifocal hepatocellular necrosis and mixed inflammatory cell infiltration were observed in the positive control group (sulfadiazine sodium), and the damage was aggravated; while no obvious granulomas and mononuclear cell infiltration were observed in the triclosan treatment group, and the liver tissue lesions were significantly improved.

[0060] Spleen: Mixed cell infiltration was observed in the blank control group; the lesions were aggravated in the positive control group, with mixed cell infiltration accompanied by necrosis and macrophage infiltration; although macrophage infiltration was still present in the triclosan treatment group, the degree of mixed cell infiltration was significantly reduced compared with the blank group and the positive control group.

[0061] Kidneys: Mixed cell infiltration was observed in both the blank group and the positive control group, with similar lesion severity; the lesion severity in the triclosan treatment group was not significantly reduced, but the location of the lesions changed;

[0062] Heart, lungs, and brain: No significant pathological changes were observed in any group.

[0063] The above results indicate that triclosan treatment can significantly reduce inflammatory lesions in the liver and spleen caused by Toxoplasma gondii infection, improve organ damage, and does not cause significant drug-related damage, demonstrating good treatment safety.

[0064] Example 3: Preparation of oral triclosan formulation

[0065] Formula: 10g triclosan, 25g microcrystalline cellulose, 60g lactose, 3g crospovidone, 1.5g magnesium stearate, making a total of 1000 tablets.

[0066] Preparation method:

[0067] Step 1: Pass triclosan, microcrystalline cellulose, and lactose through a 100-mesh sieve and mix thoroughly;

[0068] Step 2: Add binder to form a soft mass, then granulate it through a 20-mesh sieve;

[0069] Step 3: Dry the wet granules at 60℃ until the moisture content is <3.0%;

[0070] Step 4: Mix the dried granules with crospovidone and magnesium stearate until uniform, and then granulate.

[0071] Step 5: Compress the tablets using a tablet press to obtain triclosan oral tablets, each containing 10mg of triclosan.

[0072] Example 4: Combined use of triclosan and sulfadiazine sodium

[0073] Experimental Objective: To investigate the combined use of triclosan with other anti-Toxoplasma gondii drugs.

[0074] Experimental plan:

[0075] Drug A: Oral administration of triclosan oral tablets prepared in Example 3, with a daily dose of 100 mg / kg;

[0076] Drug B: Oral sulfadiazine sodium tablets, daily dose is 50 mg / kg;

[0077] Usage: For individuals infected with Toxoplasma gondii, administer therapeutically effective doses of drug A and drug B simultaneously or sequentially daily for 5-7 consecutive days.

[0078] Expected results: The combination therapy is expected to have a synergistic or additive insecticidal effect, which may help reduce the dosage of each drug, thereby reducing potential toxic side effects and delaying the development of Toxoplasma gondii resistance.

[0079] Conclusion: The above examples fully demonstrate the specific application of triclosan in the preparation of drugs for treating toxoplasmosis. It not only effectively inhibits the proliferation of Toxoplasma gondii and improves the survival rate of infected animal models, but also significantly reduces pathological damage to multiple organs and protects the central nervous system. It can be formulated into oral dosage forms using conventional pharmaceutical methods and can also be used in combination with other drugs.

[0080] In summary, triclosan, as a candidate drug against Toxoplasma gondii, exhibits excellent comprehensive benefits: it strongly inhibits the parasite in vitro, effectively protects the host, improves survival rate, and reduces multi-organ pathological damage in vivo, while also demonstrating good safety. Triclosan can be used to prepare safe and effective anti-Toxoplasmosis drugs, showing significant advantages, particularly in improving host survival rate, reducing parasite burden, and alleviating multi-organ pathological damage, thus demonstrating promising development and application prospects.

[0081] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The application of the compound triclosan in the preparation of drugs for treating toxoplasmosis.

2. The use of the compound triclosan according to claim 1 in the preparation of a drug for treating toxoplasmosis, characterized in that, The drug is used to inhibit the proliferation of Toxoplasma gondii.

3. The use of the compound triclosan according to claim 1 in the preparation of a drug for treating toxoplasmosis, characterized in that, The drug is used to alleviate pathological damage to one or more organs caused by Toxoplasma gondii infection, including the heart, liver, spleen, kidneys, and brain.

4. The use of the compound triclosan according to claim 1 in the preparation of a drug for treating toxoplasmosis, characterized in that, The drug is used to protect brain tissue or to penetrate the blood-brain barrier to treat toxoplasmosis infection of the central nervous system.

5. The use of the compound triclosan according to claim 1 in the preparation of a medicament for treating toxoplasmosis, characterized in that, The drug contains a therapeutically effective amount of triclosan and a pharmaceutically acceptable carrier or excipient.

6. The use of the compound triclosan according to claim 1 in the preparation of a drug for treating toxoplasmosis, characterized in that, The drug is an oral formulation.

7. The use of the compound triclosan according to claim 1 in the preparation of a drug for treating toxoplasmosis, characterized in that, When administered to an individual, the daily dose of triclosan is 50 mg / kg to 200 mg / kg body weight.

8. The use of the compound triclosan according to claim 1 in the preparation of a medicament for treating toxoplasmosis, characterized in that, The drug is used in combination with at least one other anti-Toxoplasma gondii drug.

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