Coccidia
The apicoplast of apicomplexan parasites is believed to be the site of action. The study suggests that the enzyme/enzyme systems targeted by Ponazuril may differ among apicomplexans, or the downstream effects of inhibition may differ. [1]

Toltrazuril sulfone prevents S. neurona merozoite development[1]. 48 hours after exposure, toltrazuril sulfone inhibits the growth of N. caninum[1]. Toltrazuril sulfone may have inhibitory effects on various apicomplexans by targeting distinct enzymes or enzyme systems[1]. Following the second division by endodyogeny, toltrazuril sulfone (5 mg/ml; 20 hours) inhibits T. gondii replication[2].

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At a concentration of 5 μg/ml, Ponazuril inhibits the in vitro development of Neospora caninum tachyzoites and Sarcocystis neurona merozoites. The inhibitory effect on N. caninum becomes apparent after approximately 48 hours of exposure. Treated tachyzoites develop vacuoles and undergo degeneration. Ponazuril also inhibits the development of S. neurona merozoites, with treated merozoites and maturing schizonts developing vacuoles and degenerating. The drug inhibits cytokinesis in S. neurona schizonts. [1]
Transmission electron microscopy (TEM) studies on N. caninum show that Ponazuril treatment (5 μg/ml) causes degeneration of tachyzoites, characterized by the presence of multiple large vacuoles in the cytoplasm. The nuclear membrane often appears swollen. While the drug interferes with normal division in a few parasites, causing the presence of multiple nuclei, this is not a frequent occurrence. [1]
TEM studies on S. neurona show that Ponazuril treatment (5 μg/ml) results in degenerating schizonts and groups of degenerating merozoites containing vacuoles. Merozoites are often observed budding from the surface of these degenerating schizonts. Occasionally, apparently viable merozoites can be seen in the same host cell as degenerating schizonts, suggesting some merozoites may complete development before the full effect of the drug is expressed on the schizont. [1]
In light microscopy studies with N. caninum in CV-1 cells, the multiplication rates of tachyzoites in control and 5 μg/ml Ponazuril-treated groups were similar for the first 24 hours. Between 24 and 48 hours, the drug began to affect tachyzoite development. By 72 hours, the distinction between individual tachyzoites was obscured in most treated cells, making quantitative counts difficult. The percentage of host cells containing more than 5 divisional cycles remained above 25% in treated cells but was never above 10% in infected controls, suggesting that host cell lysis was not occurring as readily due to the presence of non-viable tachyzoites. Similar results were obtained in Hs68 cells. [1]
Light microscopic studies with Ponazuril and S. neurona in CV-1 cells were inconclusive, with no clear visible difference between the development of treated and control schizonts. [1]
The study notes that Ponazuril at doses higher than 5 μg/ml induced changes in CV-1 host cells, limiting the ability to examine higher doses. [1]

For ten days, toltrazuril sulfone (10–20 mg/kg; p.o.) is an effective treatment for both prevention and treatment of toxoplasmosis in mice[2].

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In outbred female CD-1 mice subcutaneously infected with 1,000 tachyzoites of the RH strain of T. gondii, orally administered Ponazuril was effective in preventing and treating acute toxoplasmosis. [2]
For prevention, mice given 10 or 20 mg/kg Ponazuril starting one day before infection and then daily for 10 days were completely protected from fatal toxoplasmosis. No relapse occurred after treatment stopped. T. gondii DNA was detected by PCR in the brain of only 1 out of 5 mice in the 10 mg/kg group and in none of the 5 mice in the 20 mg/kg group. [2]
For treatment of established infection, 100% of mice treated with 10 or 20 mg/kg Ponazuril starting 3 days after infection and then daily for 10 days survived. PCR detected T. gondii DNA in the brains of 1 out of 4 mice tested in the 10 mg/kg group and in all 5 mice in the 20 mg/kg group. [2]
When treatment was delayed until 6 days after infection, 60% of mice treated with 10 mg/kg Ponazuril survived. In contrast, 100% of mice treated with 20 mg/kg or 50 mg/kg (excluding one mouse that died of aspiration pneumonia) survived. All surviving mice in these groups were positive for T. gondii by PCR on brain tissue. No relapse occurred after treatment stopped. [2]

For light microscopy studies, CV-1 cells (or Hs68 cells for N. caninum comparisons) were grown on coverslips in 6-well plates and infected with 2×10⁵ N. caninum tachyzoites or an unspecified number of S. neurona merozoites. After a 3-hour incubation to allow parasite entry, the medium was replaced with maintenance medium (RPMI 1640 with 2% fetal calf serum and antibiotics) with or without 5 μg/ml Ponazuril. Ponazuril was first dissolved in DMSO to make a 1 mg/ml stock solution. Coverslips were removed at various time points (13, 24, 48, 72, and 94 hours post-treatment), fixed in buffered formalin, placed in methanol, and then stained with Diff-Quick stain. For N. caninum, the number of parasites in 100 infected host cells was counted at each time point, and the number of divisions was calculated based on the number of tachyzoites per vacuole. For S. neurona, the numbers of immature and mature schizonts in the first 100 infected cells were recorded. [1]
For transmission electron microscopy (TEM) studies, CV-1 cells (or Hs68 cells for N. caninum comparisons) in 25-cm² flasks were infected with 2×10⁶ tachyzoites of N. caninum or merozoites of S. neurona for 3 hours. The medium was then replaced with control media or media containing 5 μg/ml Ponazuril. On days 5-9 post-treatment, flasks were scraped, and the pelleted cells were fixed in 3% glutaraldehyde, post-fixed in 1% osmium tetroxide, dehydrated in an ethanol series, and embedded in resin. Thin sections were stained with uranyl acetate and lead citrate and examined with a transmission electron microscope. [1]

Animal Model: Female CD-1 mice[2]
Dosage: 10 mg/kg, 20 mg/kg
Administration: Oral administration, daily, for 10 days
Result: Prevented and protected mice from toxoplasmosis.

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The in vivo efficacy of Ponazuril was tested in outbred female CD-1 mice. A suspension of Ponazuril (50 mg/ml) was diluted in distilled water and administered orally. All mice were infected subcutaneously in the dorsal scapular region with 1 × 10³ tachyzoites of the RH strain of *T. gondii*. [2]
For the prevention study, mice (5 per group) were treated daily for 10 days with 10 or 20 mg/kg Ponazuril, starting one day before infection. For the treatment study, mice (5 per group) were treated daily for 10 days with 10, 20, or 50 mg/kg Ponazuril, starting 3 or 6 days after infection. Control groups received distilled water. Mice were weighed daily and monitored for mortality. At 8 weeks post-infection, surviving mice were bled, and serum was tested for antibodies to *T. gondii* using a modified direct agglutination test (MAT). Brains were collected for PCR analysis to detect *T. gondii* DNA. [2]

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The in vivo efficacy of Ponazuril was tested in outbred female CD-1 mice. A suspension of Ponazuril (50 mg/ml) was diluted in distilled water and administered orally. All mice were infected subcutaneously in the dorsal scapular region with 1 × 10³ tachyzoites of the RH strain of T. gondii. [2]
For the prevention study, mice (5 per group) were treated daily for 10 days with 10 or 20 mg/kg Ponazuril, starting one day before infection. For the treatment study, mice (5 per group) were treated daily for 10 days with 10, 20, or 50 mg/kg Ponazuril, starting 3 or 6 days after infection. Control groups received distilled water. Mice were weighed daily and monitored for mortality. At 8 weeks post-infection, surviving mice were bled, and serum was tested for antibodies to T. gondii using a modified direct agglutination test (MAT). Brains were collected for PCR analysis to detect T. gondii DNA. [2]

Ponazuril is identified as a major metabolite of the anticoccidial drug toltrazuril. [1]

The study notes that Ponazuril at doses higher than 5 μg/ml induced changes in CV-1 host cells in culture, preventing the examination of its effects on parasites at those higher concentrations. [1]

[1]. The effects of ponazuril on development of apicomplexans in vitro. J Eukaryot Microbiol. May-Jun 2005;52(3):231-5.

[2]. Efficacy of ponazuril in vitro and in preventing and treating Toxoplasma gondii infections in mice. J Parasitol. 2004 Jun;90(3):639-42.

Ponazuril, sold by Bayer under the brand name Marquis, is a drug currently approved for the treatment of equine protozoan spinal encephalitis caused by Sarcocystis neurona.

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Ponazuril is a triazine anticoccidial drug used to treat equine protozoal myeloencephalitis (EPM), a major neurological syndrome of horses caused by Sarcocystis neurona. It is the major metabolite of toltrazuril, another anticoccidial drug. This study was conducted to better understand its mode of action against tissue cyst-forming coccidia, specifically comparing its effects on Neospora caninum (which divides by endodyogeny) and Sarcocystis neurona (which divides by endopolygeny). The study hypothesized that the drug's effects would be similar for parasites dividing by the same mechanism (endodyogeny for T. gondii and N. caninum) but found that Ponazuril affects these two parasites differently. It induces multinucleate stage formation and inhibits cytokinesis in T. gondii, but causes more direct degeneration of N. caninum tachyzoites without frequent multinucleate stage formation. The effects on S. neurona include both degeneration and inhibition of cytokinesis. The drug is believed to act on the apicoplast, and the different phenotypic effects observed may be due to differences in the targeted enzyme systems or downstream pathways among these apicomplexan parasites. The study suggests that at the 5 μg/ml dose, Ponazuril may be static (inhibitory) rather than completely cidal (lethal) for some parasite stages, but that static agents in vitro can be cidal in vivo with assistance from the host's immune system. [1]

457.056

C, 47.27; H, 3.09; F, 12.46; N, 9.19; O, 20.99; S, 7.01

69004-04-2

Toltrazuril;69004-03-1

3050408

Solid powder

1.507g/cm3

242°

1.572

2.699

1

9

4

31

836

0

VBUNOIXRZNJNAD-UHFFFAOYSA-N

InChI=1S/C18H14F3N3O6S/c1-10-9-11(24-16(26)22-15(25)23(2)17(24)27)3-8-14(10)30-12-4-6-13(7-5-12)31(28,29)18(19,20)21/h3-9H,1-2H3,(H,22,25,26)

1-Methyl-3-(4-(p-((trifluoromethyl)sulfonyl)phenoxy)-m-tolyl)-s-triazine-2,4,6(1H,3H,5H)-trione.

BAY-Vi 9143; BAY-Vi-9143; BAY-Vi9143; Ponazuril; Toltrazuril sulfone; Marquis.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~109.32 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.47 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.47 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.47 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)