Application of benzoylmetronidazole in preparation of toxoplasma gondii resisting medicine

Abstract

The invention belongs to the technical field of medicines, and particularly relates to novel medical application of benzoylmetronidazole, namely application of benzoylmetronidazole in preparation of toxoplasma gondii resisting medicines. The invention particularly relates to application of benzoylmetronidazole serving as an active ingredient in preparation of drugs for preventing and / or treating toxoplasma infection.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of benzoyl metronidazole in the preparation of drugs against Toxoplasma gondii. Background Technology

[0002] Toxoplasmosis is a zoonotic parasitic disease caused by *Toxoplasma gondii*. Infected individuals are often asymptomatic, but in immunocompromised individuals (such as HIV patients and organ transplant recipients) and fetuses with congenital infection, it can lead to serious consequences, including encephalitis, retinochoroiditis, and even death. Currently, the combination of sulfonamides (such as sulfadiazine) and pyrimethamine is one of the standard clinical treatments for toxoplasmosis. However, this existing treatment regimen has significant limitations. First, long-term use can easily cause toxic side effects such as bone marrow suppression, allergic reactions, and kidney crystals, resulting in poor patient tolerance. Second, existing literature reports that *Toxoplasma gondii* strains have developed resistance to existing drugs, leading to decreased treatment efficacy. Therefore, developing novel, highly effective, and safe anti-toxoplasmosis drugs has become an urgent technical problem to be solved in this field. Summary of the Invention

[0003] This invention reveals that metronidazole benzoylmethionine exhibits significantly superior anti-Toxoplasma gondii effects compared to the control drug sulfadiazine sodium in both in vitro and in vivo models, while also demonstrating good safety. This provides a novel solution to overcome the shortcomings of existing technologies.

[0004] To address the shortcomings of existing technologies, this invention is implemented through the following solution:

[0005] This invention provides the use of benzoyl metronidazole in the preparation of drugs against Toxoplasma gondii.

[0006] The present invention also provides the use of benzoyl metronidazole in the preparation of drugs that inhibit the proliferation of intracellular Toxoplasma gondii.

[0007] The present invention also provides the use of benzoyl metronidazole in the preparation of drugs with in vitro anti-Toxoplasma gondii activity.

[0008] This invention also provides the use of benzoyl metronidazole in the preparation of short-term protective drugs against Toxoplasma gondii.

[0009] The present invention also provides the use of benzoyl metronidazole in the preparation of drugs for inhibiting visceral infections in mammals.

[0010] The present invention also provides the use of benzoyl metronidazole in the preparation of medicaments for relieving symptoms of inflammatory cell infiltration in viscera.

[0011] In this invention, when the anti-Toxoplasma gondii drug is used to treat Toxoplasma gondii infection in mammals, the dosage of metronidazole is ≤50 mg / kg. In the actual operation of this invention, it was found that the maximum safe concentration of metronidazole is 50 μg / mL, while a dose of 25 mg / kg can significantly improve the survival rate of mice.

[0012] In this invention, the drug comprises benzoyl metronidazole and its pharmaceutically acceptable salt, organic matter, or any pharmaceutically acceptable excipient.

[0013] In this invention, the dosage form of the drug includes any one of tablets, capsules, oral liquids, sprays, or injections.

[0014] Compared with existing technologies, the present invention has the following advantages:

[0015] This invention relates to the application of metronidazole in the preparation of anti-Toxoplasma gondii drugs. Systematic in vitro and in vivo experiments have demonstrated that metronidazole possesses significant anti-Toxoplasma gondii activity and good safety. In vitro experiments showed that the maximum safe concentration of metronidazole in Vero cells was 50 μg / mL, at which concentration, the inhibition rate against Toxoplasma gondii reached 14.3%, and the inhibitory effect was dose-dependent, superior to the control drug sulfadiazine sodium. Plaque pheromone plaque assays further confirmed its excellent in vitro antiparasitic effect. In vivo mouse model results showed that metronidazole at a dose of 25 mg / kg significantly improved the survival rate of infected mice (30%), and its short-term protective effect was stronger than that of the sulfadiazine sodium control group. This dose effectively controlled the amount of Toxoplasma gondii in ascites, and showed an antiparasitic effect superior to or equivalent to the control group on days 1 and 7. Organ infection assessment showed that metronidazole could inhibit or reduce Toxoplasma gondii infection of the heart, liver, and kidneys. Histopathological observations showed that the drug significantly improved inflammatory cell infiltration in the spleen and kidneys, promoted the recovery of pathological damage, and did not cause significant drug-related harm, demonstrating good safety. As an anti-Toxoplasma gondii drug candidate, benzoyl metronidazole shows clear therapeutic potential and application prospects. Attached Figure Description

[0016] Figure 1 For cytotoxicity testing;

[0017] in Figure 1 A represents the maximum safe concentration of benzoyl metronidazole. Figure 1 B represents the maximum safe concentration of sulfadiazine sodium;

[0018] Figure 2 To determine the in vitro anti-Toxoplasma gondii effect of benzoyl metronidazole using the CCK-8 assay;

[0019] in Figure 2A indicates that the inhibitory effect of benzoyl metronidazole on the proliferation of intracellular Toxoplasma gondii is dose-dependent. At the maximum safe concentration of 50 μg / mL, benzoyl metronidazole can significantly inhibit the proliferation of intracellular Toxoplasma gondii, with an inhibition rate of 14.3%. Figure 2 B represents the highest inhibition rate of sulfadiazine sodium at 12.5 μg / mL, which is 1.6%.

[0020] Figure 3 To detect the in vitro anti-Toxoplasma gondii effect of benzoyl metronidazole using the plaque assay;

[0021] Figure 4 The survival time curve of mice after treatment with metronidazole benzoyl;

[0022] Figure 5 The amount of *Helicobacter pylori* in the ascites of mice after treatment with metronidazole benzoyl;

[0023] Figure 6 To detect organ infection status in mice using PCR;

[0024] Figure 7 Histopathological observation of mice in each group. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0026] Example

[0027] 1. Cell resuscitation and passage

[0028] Resuscitated Vero cells were resuspended in DMEM medium containing 10% fetal bovine serum (FBS) and then cultured in a 37°C, 5% CO2 incubator. Once approximately 90% dense monolayer of cells had formed at the bottom of the flask, the original medium was discarded, and 1 mL of trypsin was added for digestion. After digestion, fresh culture medium was added to terminate the reaction, and the cell suspension was mixed and transferred to a new culture flask for continued culture.

[0029] 2. Resuscitation and propagation of insect strains

[0030] The *Toxoplasma gondii* strain (F11 generation) was removed from liquid nitrogen and resuscitated. It was then resuspended in 1 mL of DMEM medium containing 1% fetal bovine serum and inoculated into confluent Vero cells. After a large amount of tachyzoites had overflowed, they were scraped from the culture flask using a cell scraper and repeatedly aspirated with a 27 G needle to disrupt the cells and fully release the tachyzoites. The cells were then centrifuged at 200 g for 5 min, and the supernatant was filtered through a 5 μm filter and centrifuged again at 800 g for 10 min. The supernatant was discarded. The strain was passaged three times. After the overflow stabilized, the cells were counted using a cell counter for subsequent experiments.

[0031] 3. Cytotoxicity test

[0032] The cytotoxic effect of metronidazole on Vero cells was evaluated using the CCK-8 assay. First, metronidazole was dissolved in DMSO to prepare a 1 g / mL stock solution. During the experiment, it was diluted to the predetermined concentration gradient using DMEM medium containing 1% FBS. Vero cells were then cultured at a concentration of 1 × 10⁶ cells / mL. 5 Cells were seeded at a density of [number] cells / mL in 96-well plates and incubated at 37°C with 5% CO2 for 12 hours until a monolayer of cells formed. The original culture medium was then discarded, and fresh culture medium containing different concentrations of the drug was added. The drug concentrations for the experimental groups were set at 1000, 800, 600, 500, 400, 200, 100, and 50 μg / mL. Wells without drug were used as a control group, and wells containing only culture medium but no cells were used as a blank group. After culturing for another 24 hours, CCK-8 reagent was added to each well, and after a 2-hour reaction, the absorbance at 450 nm was measured using a multi-mode 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 drug concentration with a cell proliferation rate greater than 90% was determined as the maximum safe concentration for subsequent in vitro screening of anti-Toxoplasma gondii activity.

[0033] 4. In vitro anti-Toxoplasma gondii test of the drug

[0034] Vero cells were seeded into 96-well plates using the same method described above, and inoculated with a suspension of Toxoplasma gondii tachyzoites at a density of 5 × 10³ cells / mL, and incubated for 8 hours. The cells were then washed twice with PBS to remove any culture medium containing parasites that had not yet invaded the cells. Fresh culture medium containing different concentrations of metronidazole benzoate was added to each well within the safe concentration range of the drug, and the cells were incubated at 37°C in a 5% CO2 incubator for 48 hours. After incubation, the supernatant was discarded, and 100 μL of fresh culture medium and 10 μL of CCK-8 solution were added to each well, followed by incubation for another 2 hours. The absorbance at 450 nm was measured using a multi-mode microplate reader, and the compound with the best anti-parasitic effect was selected for subsequent experiments. The relative inhibition rate (%) was calculated using the following formula: (OD control group – OD drug-treated group) / OD control group × 100%.

[0035] 5. Spot test

[0036] Vero cells were loaded at 2 × 10 5 Inoculated at a density of *Toxoplasma gondii* cells / mL into 6-well plates and incubated at 37°C with 5% CO2 until a monolayer was formed. An equal volume of *Toxoplasma gondii* tachyzoites was then inoculated, and incubation continued for 8 hours. The culture medium was then discarded, and the plates were washed twice with PBS to remove any uninoculated cells. Next, 200 μg / mL of metronidazole benzoate (experimental group) and sulfadiazine sodium (control group) were added, with a blank control group receiving no drugs. Each group was incubated for 24, 48, and 72 hours, respectively. After each time point, the culture medium was discarded, the plates were washed twice with PBS, stained with Giemsa stain for 10 minutes, washed twice more with PBS, and finally observed and photographed under an inverted microscope (×40).

[0037] 6. In vivo anti-Toxoplasma gondii test in mice

[0038] The mice used in the experiment were randomly divided into 5 groups, with 10 mice in each group. All mice were intraperitoneally injected with 1×10⁻⁶ mmol / L. 4 The dose was 1 / mL of tachyzoites. Administration began 4 hours after inoculation, once daily by gavage for 5 consecutive days. Grouping and administration regimens were as follows: metronidazole was administered in three dose groups: high (50 mg / kg), medium (25 mg / kg), and low (12.5 mg / kg); sulfadiazine sodium (50 mg / kg) served as the control group; both groups used corn oil as a solvent; a blank control group was also included, which received only an equal volume of corn oil by gavage. The therapeutic effects of each drug on Toxoplasma gondii infection in mice were observed within one week after the end of administration.

[0039] 7. Survival rate and ascites worm count detection

[0040] During the 7-day observation period, the survival of mice in each group was closely monitored daily. On days 1, 3, 5, and 7 post-infection, one mouse from each group was randomly selected, and ascites fluid samples were collected. The number of Toxoplasma gondii in the ascites fluid was counted under a microscope using a hemocytometer. The survival rate (%) was calculated as: (Number of surviving mice at the end of the experiment / Total number of mice in each group) × 100%.

[0041] 8. PCR method for detecting the therapeutic effects on mouse organs

[0042] To evaluate the therapeutic effect of metronidazole benzoyl peroxide on Toxoplasma gondii infection in different organs of mice, DNA was extracted from mouse heart, liver, spleen, lung, kidney, and brain tissues and detected by PCR. The total reaction volume was 25 μL, including 12.5 μL Taq Master Mix, 1 μL each of forward and reverse primers, 1 μL template DNA, and 9.5 μL ddH2O. The PCR program was as follows: 95℃ pre-denaturation for 5 minutes; followed by 35 cycles, including 95℃ denaturation for 10 seconds, 60℃ annealing for 10 seconds, and 72℃ extension for 20 seconds; and finally, extension at 72℃ for 5 minutes. The amplified products were analyzed by 2% agarose gel electrophoresis.

[0043] The forward primer is shown in SEQ ID NO.1: 5′-GGAACTGCATCCGTTCATGAG-3′;

[0044] The reverse primer is shown in SEQ ID NO.2: 5′-TCTTTAAAGCGTTCGTGGTC-3′.

[0045] The gene being tested is the B1 gene.

[0046] 9. Pathological changes in mouse organs

[0047] On day 7 of infection, organs such as the heart, liver, spleen, lungs, kidneys, and brain of mice from each group were collected and fixed in 4% paraformaldehyde for one week. Subsequently, the fixed tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). Finally, the histopathological changes were observed and analyzed under an optical microscope (×200x).

[0048] Application examples

[0049] 1. Cytotoxicity test

[0050] To evaluate the toxic effects of benzoyl metronidazole and the control drug on Vero cells, this study used the CCK-8 assay. The results showed that the maximum safe concentration of benzoyl metronidazole was 50 μg / mL. Figure 1 A), while the maximum safe concentration of sulfadiazine sodium in the control group was 1250 μg / mL (A). Figure 1B). Based on the above safe concentration range, subsequent in vitro anti-Toxoplasma gondii experiments were conducted.

[0051] 2. In vitro anti-Toxoplasma gondii test of the drug

[0052] The inhibitory effect of metronidazole on Toxoplasma gondii was evaluated using the CCK-8 assay, and the results are as follows: Figure 2 As shown. At the maximum safe concentration of 50 μg / mL, metronidazole significantly inhibited the proliferation of Toxoplasma gondii within cells, with an inhibition rate of 14.3%, and this inhibitory effect increased in a dose-dependent manner. Figure 2 A). The inhibitory effect on Toxoplasma gondii became more pronounced with increasing drug concentration. The highest inhibition rate of the positive control drug, sulfadiazine sodium, at 12.5 μg / mL was 1.6% ( Figure 2 B). Compared with the control group, metronidazole benzoyl showed superior anti-insect activity in vitro, indicating its strong potential for in vitro anti-Toxoplasma gondii activity.

[0053] 3. Plaque test

[0054] To further evaluate the in vitro anti-Toxoplasma gondii activity of benzoyl metronidazole, we performed a plaque affinity assay. Figure 3 As shown, after 72 hours of treatment at the same concentration of 200 μg / mL, the number of Toxoplasma gondii observed in the benzoyl metronidazole group (3A) was the lowest compared with the sulfadiazine sodium control group (3B) and the blank control group (3C), indicating that it had the best insecticidal effect.

[0055] 4. In vivo anti-Toxoplasma gondii test in mice

[0056] In vitro experiments showed that metronidazole benzoyl (MBZ) has significant antiparasitic activity. To further evaluate its in vivo effects, we conducted an experiment in mice infected with Toxoplasma gondii RH tachyzoites via intraperitoneal infection. The results showed (Table 1) that, compared with the control group (PC), metronidazole benzoyl at a dose of 25 mg / kg significantly improved the survival rate of mice (30%). The survival rates of the high-dose group (50 mg / kg) and the low-dose group (12.5 mg / kg) were both 20%, comparable to the control group (20%), but superior to the blank group (10%). Furthermore, this drug not only reduced the morbidity rate in mice but also improved their survival rate. Figure 4 In summary, at a dose of 25 mg / kg, metronidazole benzoyl was more effective than sodium sulfadiazine in providing short-term protection to mice.

[0057] Regarding the changes in the number of tachyzoites in peritoneal fluid ( Figure 5As treatment duration increased, the number of *Helicobacter pylori* worms in the ascites fluid of mice showed different trends. During the 7-day observation period, the high-dose benzoyl metronidazole group (50 mg / kg) consistently demonstrated excellent antiparasitic effects. The medium-dose group (25 mg / kg) showed superior or equivalent antiparasitic effects to the sulfadiazine sodium control group (PC, 50 mg / kg) on ​​both days 1 and 7. Therefore, benzoyl metronidazole at a dose of 25 mg / kg provides better short-term protection in mice than sulfadiazine sodium.

[0058] Table 1. In vivo efficacy of the drug against Toxoplasma gondii

[0059] 5. PCR method for detecting worm bearing in mouse organ tissues

[0060] After 7 days of treatment with a medium dose of metronidazole, mice in each group had heart, liver, spleen, lung, kidney, and brain tissues collected. DNA was extracted and PCR was performed using primers specific to the Toxoplasma gondii B1 gene to assess the infection status of Toxoplasma gondii in each organ. PCR amplification results ( Figure 6 The results showed that Toxoplasma gondii DNA was still detectable in the spleen, lungs, and brain tissue, but not in the heart, liver, and kidney tissues. Compared to the control group, the band brightness in positive tissues was reduced. These results indicate that metronidazole can inhibit or alleviate Toxoplasma gondii infection in some organs of mice, including the heart, liver, and kidneys.

[0061] 6. Pathological changes in mouse organs

[0062] On day 7 of treatment with metronidazole for Toxoplasma gondii infection, pathological observations were performed on the heart, liver, spleen, lungs, kidneys, and brain tissue of mice. The results showed that no obvious pathological changes were observed in the heart, lungs, and brain tissue of mice in any group.

[0063] In the liver, the blank control group showed microgranulomas or granulomas (red arrows), glycogen accumulation in central lobular hepatocytes (blue arrows), and mononuclear cell infiltration in the portal areas (green arrows). After drug treatment, the above lesions in the control group were slightly alleviated, but microgranulomas and mononuclear cell infiltration remained. After treatment with benzoyl metronidazole, the microgranulomas or granulomas were reduced, and the degree of mononuclear cell infiltration decreased significantly.

[0064] In the control group, the spleen showed mixed cellular infiltration (red arrows, mainly neutrophils and monocytes) and small lesions (green arrows). Mixed cellular infiltration, accompanied by some cell necrosis and apoptosis, was observed, which may be related to the side effects of the drugs used in the control group. In contrast, the mixed cellular infiltration in the spleen of the metronidazole benzoyl peroxide group completely disappeared, with no cell apoptosis or necrosis observed.

[0065] In the kidney blank group, mixed cellular infiltration was observed around the arteries and veins at the renal hilum (red arrows, mainly monocytes and lymphocytes), while the control group still showed mixed cellular infiltration around the arteries, and the condition did not improve. In the benzoyl metronidazole group, no mixed cellular infiltration was observed in the renal capsule, renal hilum, and parenchyma, and the pathological improvement was significant.

[0066] Treatment with benzoyl metronidazole can significantly reduce inflammatory cell infiltration in the spleen and kidneys, improve pathological damage in related organs, and does not cause significant drug-related damage, demonstrating good safety.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Use of benznidazole in the preparation of a drug for combating Toxoplasma gondii.

2. Use of benznidazole in the preparation of a drug for inhibiting the proliferation of intracellular Toxoplasma gondii.

3. Use of benznidazole in the preparation of a drug for combating Toxoplasma gondii in vitro.

4. Use of benznidazole in the preparation of a drug for short-term protection against Toxoplasma gondii.

5. Use of benznidazole in the preparation of a drug for inhibiting visceral infection in a mammal.

6. Use of benznidazole in the preparation of a drug for relieving the symptoms of visceral inflammatory cell infiltration.

7. The use according to any one of claims 1 to 6, characterized in that, When the drug is used to treat Toxoplasma gondii infection in a mammal, the dosage of benznidazole is ≤ 50 mg / kg.

8. The use according to any one of claims 1 to 6, characterized in that, When the drug is used to treat Toxoplasma gondii infection in a mammal, the dosage of benznidazole is 25 mg / kg.

9. The use according to any one of claims 1 to 6, characterized in that, The drug comprises benznidazole and pharmaceutically acceptable salts, organic substances or pharmaceutically acceptable adjuvants thereof.

10. The use according to any one of claims 1 to 6, characterized in that, The dosage form of the drug includes any one of tablets, capsules, oral solutions, sprays or injections.

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