In vitro action of antiparasitic drugs , especially artesunate , against Toxoplasma gondii Ação in vitro de drogas antiparasitárias , especialmente artesunato , contra Toxoplasma gondii

Introduction: Toxoplasmosis is usually a benign infection, except in the event of ocular, central nervous system (CNS), or congenital disease and particularly when the patient is immunocompromised. Treatment consists of drugs that frequently cause adverse effects; thus, newer, more effective drugs are needed. In this study, the possible activity of artesunate, a drug successfully being used for the treatment of malaria, on Toxoplasma gondii growth in cell culture is evaluated and compared with the action of drugs that are already being used against this parasite. Methods: LLC-MK2 cells were cultivated in RPMI medium, kept in disposable plastic bottles, and incubated at 36oC with 5% CO2. Tachyzoites of the RH strain were used. The following drugs were tested: artesunate, cotrimoxazole, pentamidine, pyrimethamine, quinine, and trimethoprim. The effects of these drugs on tachyzoites and LLC-MK2 cells were analyzed using nonlinear regression analysis with Prism 3.0 software. Results: Artesunate showed a mean tachyzoite inhibitory concentration (IC50) of 0.075μM and an LLC MK2 toxicity of 2.003μM. Pyrimethamine was effective at an IC50 of 0.482μM and a toxicity of 11.178μM. Trimethoprim alone was effective against the in vitro parasite. Cotrimoxazole also was effective against the parasite but at higher concentrations than those observed for artesunate and pyrimethamine. Pentamidine and quinine had no inhibitory effect over tachyzoites. Conclusions: Artesunate is proven in vitro to be a useful alternative for the treatment of toxoplasmosis, implying a subsequent in vivo effect and suggesting the mechanism of this drug against the parasite.

Toxoplasmosis is a highly prevalent cosmopolitan infection, but the disease occurs in only a fraction of infected people, mainly as a nonspecific immune activation syndrome, chronically in ocular forms as chorioretinitis.The main problems are congenital disease and the infection of immunocompromised people, especially those with acquired immunodeficiency syndrome (AIDS) or those undergoing chemotherapy for cancer or transplant rejection 1 .

The etiological agent of toxoplasmosis is
Toxoplasma gondii, the development of which has many forms.Tachyzoites are found in the acute phase of the disease and are responsible for clinical manifestations.They are susceptible to the immune response of the host and to drug action.Cysts are the resistant form of the parasite, persisting for the host's entire life.Cyst walls are resistant to both drugs and the immune system 2 .
Felids are the definitive hosts for the parasite, with other mammalians and birds acting as intermediate hosts.Humans can be infected either congenitally or through ingestion of raw or undercooked meat; manipulation of infected meat containing tissue cysts; or consumption of water, fresh vegetables, or other food contaminated by oocysts eliminated in cat feces 1,2 .
The most effective treatment against toxoplasmosis is a combination of the drugs sulfadiazine and pyrimethamine, which can cause hematological effects that are controlled with the administration of folinic acid.An association of great interest is the one between trimethoprim and sulfamethoxazole.Known as cotrimoxazole, its active compounds act synergistically, inhibiting two consecutive steps of folinic acid biosynthesis in a manner similar to that observed for pyrimethamine-sulfadoxine. Cotrimoxazole is well tolerated and less toxic to hematopoiesis.Human immunodeficiency virus-acquired immunodeficiency syndrome (HIV-AIDS) patients taking cotrimoxazole show a high incidence of adverse effects, and its use is discouraged in pregnant women because it crosses the placental barrier 3 .

Gomes TC et al -Artesunate against Toxoplasma gondii
Although antifolate compounds, such as pyrimethamine, exhibit good anti-Toxoplasma activity, their toxicity limits widespread use, particularly for extended treatment periods.The discovery of viable low-toxicity compounds capable of preventing and treating T. gondii would represent a great advance in the treatment of infections in immunocompromised patients.Some compounds that are effective against species of Plasmodium could be effective against T. gondii.Those agents were selected for further testing in the present study because malaria and toxoplasmosis are caused by protozoans belonging to the phylum Apicomplexa, and antimicrobial agents that have been effective for the treatment of malaria, such as artemisinin and its derivatives, also have been effective for the treatment of toxoplasmosis.Artemisinin (qinghaosu) is a product extracted from the plant Artemisia annua L. Despite the fact that artemisinin has produced teratogenic effects in laboratory animals, precluding its use in pregnant women, few adverse effects have been observed in humans 4,5 .
In the present study, the toxicity of artesunate and its effectiveness for the treatment of toxoplasmosis were studied in vitro and compared with the actions of three drugs: pyrimethamine, trimethoprim, and cotrimoxazole, which are currently in use against toxoplasmosis.Pentamidine and quinine, used for the treatment of other protozoans, also were evaluated and compared.Pentamidine is an organic compound and derivative of guanidine that has shown activity against Leishmania sp., African trypanosomiasis, and pneumonia caused by Pneumocystis carinii (jiroveci).Quinine is an alkaloid extracted from species of the genus Cinchona, the application of which is limited to cases of malaria caused by Plasmodium falciparum.

Parasites
Tachyzoites of the type I RH strain of T. gondii were routinely maintained by intraperitoneal passage in BALB/c mice.

Drugs
All drugs were obtained from commercial sources (Sigma, USA) or as human use drugs, supplied by the pharmacy of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP).Artesunate and quinine dichlorohydrate were supplied by Cipla Medpro (Belville, SA), pentamidine was supplied by Itaca Labs (Rio de Janeiro, BR), and cotrimoxazole was supplied by Ducto (Anapolis, BR).

Cell culture
The epithelial cell line LLC-MK2, derived from rhesus monkey (Macaca mulatta) kidneys, was used.Cells were cultivated in RPMI medium with the addition of 10% inactivated bovine fetal serum and gentamicin.Cultures were kept in disposable plastic bottles and incubated at 36ºC with 5% CO 2 .

In vitro assays of drug effectiveness and toxicity
The assays were conducted in four stages, the first three on consecutive days.On the first day, LLC-MK2 cells were extracted from a plastic bottle with ATV enzyme and counted in a Neubauer counting chamber.Cells were diluted in RPMI medium containing 10% inactivated bovine fetal serum until a concentration of 1 × 10 4 was obtained.One hundred microliters of the mixture was added to each well of a 96-well plate and placed inside the CO 2 incubator.On the second day, the supernatant from all the wells was aspirated.Tachyzoites from the peritoneal fluid of BALB/c mice were extracted with a syringe and diluted in complete RPMI medium containing 10% bovine fetal serum until a concentration of 1 × 10 4 was obtained.One hundred microliters of the mixture was added to the wells in rows A to E and columns 1 to 12 of the plate and put into a CO 2 incubator.The remaining wells were filled with complete RPMI containing 10% bovine fetal serum.On the third day, the plate was washed with complete RPMI medium containing 10% inactivated bovine fetal serum.A mother solution of each drug was prepared by diluting each one in a solution of complete RPMI medium containing 10% inactivated bovine fetal serum until a 200μL/mL concentration was obtained.Two-fold serial dilutions of all compounds were performed, starting with initial concentrations of 100μg/mL.Therefore, the concentrations used were as follows: 100, 50, 25, 12.5, 6.25, 3.1, 1.6, 0.8, 0.4, 0.2, and 0.1μg/mL.For pyrimethamine and trimethoprim, the first dilution was made in dimethyl sulfoxide, and the remaining dilutions were made in RPMI because of the low solubility of these compounds in water.On another 96-well plate, 200μL of each drug solution was pipetted into wells in column 1, whereas 100μL of RPMI medium containing 10% inactivated bovine fetal serum solution was pipetted into the wells in columns 2 to 12.A two-fold serial dilution was performed by transferring 100μL from column 1 to those in column 2 and repeating this procedure until column 11.The contents of the second plate were transferred to the first plate and placed inside the CO 2 incubator.The reaction was interrupted when the tachyzoites had destroyed all the cells from the positive control wells.After that, the supernatant was moved, and the plate was washed with PBS, fixed with methanol, and stained with 1% aqueous crystal violet solution.After being washed, the plate was dried, and 200µL of methanol was added to dissolve the stain.The A 620 was measured with an ELISA microplate reader.Adherent live cells were stained.The A 620 is proportional to the number of viable cells.This allowed both the measurement of T. gondii cell destruction, or cell toxicity, and detection of infected or non-infected cell layers.

Statistical analysis
Drug effects on both tachyzoites in vitro and LLC-MK2 cells were analyzed using nonlinear regression analysis with Prism 3.0 software, yielding mean inhibitory concentrations (IC 50 ) for the studied compounds.
Artesunate at a concentration of 100µg/mL killed all cells in the culture.Therefore, the experiment was performed again with a maximum concentration of 10µg/mL.It was observed that artesunate was effective against tachyzoites at an IC 50 of 0.075µM, resulting in preservation of the cell line (Figure 1A and 1B).The mean toxicity of the drug was 2.003µM (Figure 2A and 2B).
Cell death also was observed with pyrimethamine at 100µg/ mL in the cell culture so that experiment also was performed again with a maximum concentration of 10µg/mL.It was observed that pyrimethamine was effective against tachyzoites at an IC 50 of 0.482µM, resulting in the preservation of the cell line.The mean toxicity of the drug was 11.178µM (Figure 3A and 3B).
Cotrimoxazole proved to be effective against tachyzoites at an IC 50 of 11.884µM, whereas trimethoprim was effective against Pentamidine had no effect on tachyzoites at the tested concentrations, resulting in destruction of the cell line.The same results were observed for quinine.The mean toxicity of pentamidine was 1,316µM, and for quinine, it was 91.030µM.
The selectivity index (SI) could be calculated only for artesunate and pyrimethamine.The SI for the first one was 26.707, and that for the latter was 23.191.
In the present study, artesunate showed the highest efficacy, followed by pyrimethamine.Moreover, artesunate presented the highest toxicity among the studied compounds, again followed by pyrimethamine.
An absence of cotrimoxazole and trimethoprim was observed, yet each showed a weaker effect than artesunate and pyrimethamine.Pentamidine and quinine had no inhibitory effects over T. gondii in this study.
The mean inhibitory concentrations and toxicities, 95% confidence intervals, and r 2 values for all drugs used in the study are shown in Table 1.The selectivity index for two of the six drugs could be calculated and also is shown.As can be seen, the most effective drugs against T. gondii were pyrimethamine and artesunate.An in vitro effect of artesunate against T. gondii was found.Previous reports had demonstrated that this compound showed action against other parasites, such as Plasmodium 6 and Fasciola 7 .Although inhibitory concentrations were higher than those used against the parasites that cause malaria, artemisinin and many of its derivatives were effective against T. gondii 5 .In this experiment, artesunate exhibited a strong effect against T. gondii tachyzoites in in vitro assays.This work represents an initial step toward future studies of the in vivo action of this compound and its effect on the cystic form of the parasite.Artesunate could be an alternative to the standard pyrimethamine-sulfonamide treatment.
Effective action of artesunate against tachyzoites was observed, and it was the highest among all studied compounds.Several studies demonstrating the action of artemisinin derivatives have been conducted, including Ke Ou-Yang et al. 8 , D' Angelo et al. 9 , and Sarciron et al. 10 .El Zawary, in 2008, studied the in vitro action of artesunate against RH strain Toxoplasma and observed a significant reduction in the viability and effect of tachyzoites exposed to drugs compared with a no-treatment control.Although the efficacy of artesunate was demonstrated both here and in El Zawary, the inhibitory concentration values for artesunate found by El Zawary were higher than those observed in the present work.However, because there is no standard methodology for drug testing in cell lines, different outcomes could result 11 .
Clark et al. 12,13 observed that artesunate is toxic to the embryos of mice, rabbits, and nonhuman primates, causing cardiovascular and skeletal problems, even death, when given at higher doses and over longer periods than recommended for the treatment of malaria 12,13 .

FIGURE 1 -FIGURE 2 -
FIGURE 1 -Morphology of LLC-MK2 cells after (A) Toxoplasma gondii infection and (B) artesunate treatment following parasite challenge.Digital images are from inverted microscope phase-contrast microscopy with 20 × objective.

FIGURE 4 -
FIGURE 4 -A) Cotrimoxazole action in cell culture infected with Toxoplasma gondii at concentrations of 100 to 0.1µg/mL.B) Trimethoprim action in cell culture infected with Toxoplasma gondii at concentrations of 100 to 0.1µg/mL.