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Artemisone and Artemiside Control Acute

and Reactivated Toxoplasmosis in a Murine
Model
Ildiko R. Dunay, Wing Chi Chan, Richard K. Haynes and L.
David Sibley
Antimicrob. Agents Chemother. 2009, 53(10):4450. DOI:
10.1128/AAC.00502-09.
Published Ahead of Print 27 July 2009.

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Louis. although artemiside was more effective. Artemisinin (Fig. due to the ability of the parasite to cross the placental barrier and cause infection in the developing fetus (21. Hong Kong SAR. standard treatments are often complicated by severe side effects. and artemether. Oct.edu. are potent and rapidly acting antimalarial drugs that are currently used widely in combination therapies to treat malaria (3). Atovaquone in combination with pyrimethamine or sulfadiazine was reported to prevent relapse of toxoplasmosis in AIDS patients (7). Louis. Vol. 41). Artemiside and artemisone treatment controlled parasite replication in vivo. 2009. the current treatments control only actively proliferating parasites but have limited activity against bradyzoites within tissue cysts and hence do not eliminate the chronic infection. it has also been associated with breakthrough failures in other cases (4. Artemisinin and its derivatives. and mice survived the acute infection. and it has been reported to have some activity against tissue cysts when administered for long periods of time (2). and a chronic phase.org/ on April 22. leading to fecal shedding of a spore-like stage known as the oocyst (9). Euclid Ave. However. 10 Artemisone and Artemiside Control Acute and Reactivated Toxoplasmosis in a Murine Model䌤 Ildiko R. 24). Institute of Molecular Technology for Drug Discovery and Synthesis. Artemisinin is a new highly potent antimalarial drug that has activity against Toxoplasma gondii in vitro. 53. dihydroartemisinin (DHA). Missouri 63110. Washington University School of Medicine. People’s Republic of China2 Received 16 April 2009/Returned for modification 27 May 2009/Accepted 6 July 2009 most common clinical manifestation of reactivated disease in AIDS patients who do not receive highly active antiretroviral therapy or antiparasitic prophylaxis (25). Open Laboratory of Chemical Biology. Artemiside and artemisone displayed better inhibition than either artemisinin or artesunate against the parasite in vitro.wustl. The acute phase generally produces only mild symptoms in immunocompetent adults and is readily controlled by the immune response (29). including artesunate (Fig. Mailing address: Department of Molecular Microbiology. Toxoplasmosis is of concern during pregnancy. Humans can become infected by ingestion of oocysts that contaminate water or tissue cysts in undercooked meat. have markedly superior in vitro antimalarial activity against chloroquine-sensitive and -resistant Plasmodium falciparum lines and also display greater activities against the rodent malaria Plasmodium berghei in vivo (40). Washington University School of Medicine. In a murine model of reactivated toxoplasmosis. 4450 Downloaded from http://aac. However. Atovaquone. Fax: (314) 286-0060. Standard therapy for toxoplasmosis in humans includes combinations of pyrimethamine plus sulfadiazine. § Present address: Department of Neuropathology. and although chemotherapy is available. p.ANTIMICROBIAL AGENTS AND CHEMOTHERAPY. All Rights Reserved. The new artemisinin derivatives artemisone and artemiside. these drugs often cannot be tolerated due to severe side effects (22). Patients at risk include those with underlying conditions. 1). MO 63110. the thiomorpholine precursor of artemisone. Haynes. If left untreated. 660 S. Kowloon.00⫹0 doi:10. gondii both in vitro and in vivo. 1) is a sesquiterpene lactone produced by the plant Artemisia annua and is used for the treatment of fevers and chills as part of traditional Chinese herbal medicine (14). These results indicate that these newer derivatives of artemisinin may have potential for treatment of toxoplasmosis. reactivation leads to severe central nervous system (CNS) complications and may result in death.1128/AAC. Euclid Ave. Breihacher Str. there is a need for the development of new therapeutics for treatment of toxoplasmosis. such as AIDS. No.1 and Department of Chemistry. In the present study. the parasite crosses the gut epithelium and disseminates throughout the body (5). Germany. the efficacy of several new artemisinin derivates was investigated for treatment of mice infected with the parasite Toxoplasma gondii.asm. The life cycle includes a sexual phase in the intestine of cats. However.00502-09 Copyright © 2009. Clear Water Bay. thus. 660 S. The enhanced efficacy of artemisone is also apparent in The protozoan parasite Toxoplasma gondii infects a large number of warm-blooded animals and is estimated to chronically infect up to one-third of the world’s human population (11). 4450–4456 0066-4804/09/$08. Freiburg D-79106.2 Richard K. immunocompromised patients are predisposed to development of reactivated toxoplasmosis due to cyst rupture and uncontrolled proliferation of tachyzoites (25).. artemisinin derivatives have previously been ineffective in vivo using a rat model of toxoplasmosis.1§ Wing Chi Chan. 䌤 Published ahead of print on 27 July 2009. prolonged treatment is necessary for immunocompromised patients (22). is also active against T. Dunay. St. Asexual development takes place in a wide range of warm-blooded vertebrates and is characterized by an acute phase.. both drugs increased survival. during which the parasite propagates rapidly as tachyzoites. The latent stage of infection is characterized by the presence of parasites within tissue cysts that form in the skeletal muscle and central nervous system (25). Toxoplasmic encephalitis is the * Corresponding author. 64. David Sibley1* Department of Molecular Microbiology. originally developed for malaria. Phone: (314) 362-8873. St. Additionally. 2014 by guest Immunocompromised patients are at risk of developing toxoplasmosis. or individuals who are therapeutically immunosuppressed due to organ transplantation or cancer chemotherapy.2 and L. Hence. typified by tissue cysts containing slow-growing forms called bradyzoites (9). E-mail: sibley@borcim. the Hong Kong University of Science and Technology. Following oral uptake. . American Society for Microbiology. University of Freiburg. Further.

15 mg/kg body weight of firefly luciferin D (Biosynth AG. artesunate. Experiments contained three animals per group that were monitored for 25 days postinfection. gondii and found that it was ineffective in controlling infection (6). although the difference in potencies between P. Parasites were maintained as tachyzoites in human foreskin fibroblast cells grown in Dulbecco’s modified Eagle’s medium with 10 mM HEPES.) injection of 106 tachyzoites in 0. MA) 8 to 10 weeks of age were used for all acute infection studies.0 (GraphPad Software Inc.2. The end product of the reaction was read as absorbance at 570 nm. parasites were inoculated in 96-well plates containing monolayers of human foreskin fibroblast cells and were treated with different compounds at concentrations ranging from 0. 8. Briefly. DHA. falciparum (27). In the present study. following treatment with mild anesthesia using isofluorene and i. Staad. we tested the antiparasitic efficacy of several new derivates of artemisinin against T. The parent compound artemisinin is effective against T. 6. The luciferase expressing the PRU-Luc-GFP type II strain (kindly provided by J.2 ml phosphate-buffered saline. Artemisinin.010 ␮M to 1. 2009 ARTEMISININ ANALOGUES INHIBIT TOXOPLASMA 4451 studies involving an Aotus monkey model for P. falciparum and T.] 10 mg/kg) every day for 8 days. 9. as well as several other new derivatives (17).0 ␮M for 72 h at 37°C. gondii was used for mouse infections. Stanford University School of Medicine. Structure diagrams of artemisinin and related compounds. Animals were imaged using a Xenogen IVIS 100 (Caliper LifeSciences. 3. Despite in vitro activity. clone 2F (ATCC 50839). 1. 2 mM glutamine. previous studies tested artemether with a rat model with concurrent infection with Pneumocystis carinii and T. gondii cultured in vitro and in murine models of acute and reactivated toxoplasmosis. sulfamide derivative. and the brains were removed and homogenized in 1. Animals were maintained in an AAALAC-approved facility overseen by the Institutional Animal Care Committee at Washington University. Boothroyd. although the 50% effective concentrations (EC50s) are ⬃50-fold higher than those reported for P. have also been shown to be more potent against T. pyrimidylpiperazine derivative. MA). after which chronic infections were confirmed by examining homogenates of the brain for tissue cysts. CA) of T. injection of 0. so we first established the dose of tachyzoites that would cause lethal infection in a majority of mice (data not shown). Control and drug-treated mice were infected by intraperitoneal (i. Downloaded from http://aac. and mean values from three independent experiments were used to estimate the EC50s and EC90s by curve fitting using nonlinear regression with a variable slope in Prism 5. and 10 ␮g/ml gentamicin. and artemisone (27). CD1 mice are relatively resistant to infection by type II strains. The mechanism of action of artemisinin and its derivatives is dependent upon the presence of the endoperoxide bridge (14). Hopkinton.0 ml of sterile phosphate-buffered saline. as described previously (27). As described previously. benzylpiperazine derivative.c. gondii. animals were imaged at 2-day intervals. artemether. and parasite growth was determined based on detection of ␤-galactosidase activity. gondii suggests that different targets may be affected in these two organisms. animals were humanely sacrificed. Mouse infections. was used for in vitro growth assays (27). For growth inhibition assays. 7. Parasite burdens. The derivatives artesunate. and counts were used to estimate the total number of cysts per brain. artemether. artemiside.asm. 44 mM sodium bicarbonate. Following treatment. 10. MATERIALS AND METHODS Parasite growth assay. which expresses bacterial ␤-galactosidase. the culture medium was removed. Wilmington. gondii strain RH (ATCC 50838).). 1. 2. 10% fetal bovine serum. 4. cysts were counted from four separate 20-␮l aliquots. . Switzerland).org/ on April 22. and survival was followed for 25 days. compounds were injected (subcutaneously [s. Homogenates were examined by bright-field or phase-contrast microscopy at ⫻40 magnification. CD1 outbred female mice (Charles River Laboratories. For bioluminescence experiments. Artemisone has greatly reduced neurotoxicity compared to the major circulating metabolite of artesunate. namely DHA.p. and as such it has a superior pharmacokinetic profile for humans (28). artemisone. and 4⬘-fluorophenyl derivative. 5. the output from luciferase imaging in vivo is not directly related to parasite numbers but rather provides a relative index of the severity of infection (30). The T. and data are expressed in relative light units. gondii in vitro. 2014 by guest FIG. 53.VOL. falciparum malaria (13). pH 7.p.

1. which previous studies have indicated has an EC50 in vitro of ⬃0. as it represents the rapidly growing type I strains that are acutely virulent in the mouse model. which succumbed to infection. with compounds (10. Effective concentrations for inhibition of T. 8.263 a Compounds correspond to the respective structures shown in Fig. Mice were infected i. MO) in the drinking water (400 mg/liter) for 3 weeks beginning 2 days after infection.to 10-week-old mice were orally infected with 20 tissue cysts of PRU-Luc-GFP T. all of the compounds inhibited parasite growth. and a variable Hill slope. and 10. The 10-amino-artemisinins artemisone. Switzerland. benzylpiperazine derivative. Alternatively.c. This result likely reflects lower burdens of tachyzoites that developed in treated animals. 3C). AGENTS CHEMOTHER. Inhibition of in vitro growth of T.156 0. St. 2. mice were treated s. C57BL/6 mice are highly susceptible to infection and develop high tissue cyst burdens in the chronic phase (37). DUNAY ET AL. Louis. Table 1). Data estimated from curve fitting shown in Fig. Effect of artemisinin derivates on reactivation of latent T. with tachyzoites of type II (PRU-Luc-GFP) T. To evaluate cyst formation in the chronic phase of the infection.271 0. rather than a direct effect on tissue cysts. mice were treated with sulfadiazine (Sigma-Aldrich. None of the compounds demonstrated overt toxicity to host cell monolayers (data not shown).17 to 0.167 0. Artemiside and artemisone showed slightly better activity in vitro compared to the other compounds. gondii infection.213 0. 2014 by guest FIG. artemiside. Artemiside. Artesunate was a gift from Knoll AG. brains were removed from chronically infected mice at 25 days postinfection. gondii line expressing Escherichia coli ␤-galactosidase to parasite numbers and is highly linear over a wide range of cell numbers (33). Inbred female gamma interferon (IFN-␥⫺/⫺) mice on a C57BL/6 background were maintained under specific pathogen-free conditions. 9.182 0. Following treatment in vitro for 72 h. mice were infected as described above. Although cyst counts were not available from control mice. compounds were injected every day for 8 days. The most potent com- TABLE 1. although further testing of these earlier stocks reveal inhibition consistent with that reported here (data not shown). compounds were dissolved in DMSO and stored at 4°C for in vivo studies. 7. we used the murine model of reactivated toxoplasmosis in IFN-␥⫺/⫺ mice. sulfamide derivative. This assay relates enzyme activity from a transgenic T.368 0.and artemisonetreated mice developed significantly lower cyst numbers in the brain compared to artemisinin-treated mice. 1. and survival was monitored. We used a derivative of the RH strain for these experiments. gondii growth in vitrob Compounda EC50 (␮M) EC90 (␮M) Artemisone Artemiside Artesunate Sulfamide derivative 4⬘-Fluorophenyl derivative Benzylpiperazine derivative Pyrimidylpiperazine derivative 0. Eight.11 to 0. To control acute infection. Artemisone was somewhat less potent than previously reported (EC50 of ⬃0. pyrimidylpiperazine derivative. 2. Artemiside. least-squares fit. and cysts were counted microscopically.4452 ANTIMICROB. Two days after discontinuation of sulfadiazine.and artemisone-treated mice were able to control parasite replication and survived. Effect of artemisinin derivates on acute infection with T. the sulfamide derivative.235 0. We tested the antimicrobial activity of the most-active compounds using an in vivo assay for acute toxoplasmosis in the mouse. Parasite growth was monitored by measurement of ␤-galactosidase activity (absorbance at 570 nm) following 72 h of incubation with the different compounds (see Materials and Methods). 60% of the artemiside-treated mice and more than 50% of the artemisone-treated mice survived the infection (Fig. 3B). pounds were artemiside and artemisone (Fig.asm. Structures of compounds used in this study are given in Fig.03 ␮M) (27). and the pyrimidylpiperazine derivative were prepared as previously described (10).21 ␮M and with EC50s ranging from 0.0 mg/kg [body weight]) every day for 8 days. To follow parasite replication. 4⬘fluorophenyl derivative. The antiparasitic activities of several new artemisinin derivates were monitored using a ␤-galactosidase assay for parasite growth in vitro (27). separate studies using sublethal doses (about 10-fold lower than those used here) resulted in cyst burdens that ranged from 200 to 300 cysts per brain (data not shown).176 0. Basel. Survival and parasite burdens were monitored at 2-day intervals for 25 days. Artemisinin was obtained from Sigma Aldrich. gondii that were obtained from the brains of chronically infected wild-type mice. 2. a procedure we subsequently determined resulted in modest loss of activity. While all control mice and most artemisinin-treated mice succumbed to infection.37 ␮M (Fig. 2B). RESULTS Testing the in vitro antiparasitic activity of new artemisinin derivates. The 4⬘-fluorophenyl derivative was prepared as described previously (12). indicating that these compounds effectively blocked replication and/or resulted in killing of parasites in vivo (Fig. confirming the antiparasitic efficacy of the compounds (Fig.org/ on April 22.108 0. 2.120 0. and parasite numbers were detected by bioluminescence imaging.213 0.8 ␮M (27).158 0. To simulate reactivation of latent infection. similar to that described previously (38). Murine model of reactivated toxoplasmosis. All control mice and the majority of artemisinin-treated mice developed high parasite numbers and died due to the uncontrolled infection (Fig. gondii in the murine model. gondii. These activities compare favorably to those of artemisinin. Table 1). 3A). based on nonlinear regression.339 0. Compounds were dissolved in dimethyl sulfoxide (DMSO) and stored at ⫺80°C in single-use aliquots until used for in vitro studies. gondii by artemisinin derivatives. This loss of potency may have resulted from freezing the stocks in DMSO and thawing prior to testing. the benzylpiperazine derivative. as described above. with similar EC50s ranging from ⬃0.p. b Downloaded from http://aac. IFN␥⫺/⫺ mice are highly susceptible to both acute (39) and chronic . Compounds.

n ⫽ 2 animals per group.c. treatment with artemiside prolonged survival in 80% of the mice. In order to test the efficacy of compounds during reaction. including artemether. n ⫽ 3 or 4 animals per group. gondii in vitro (27). when the drugs were discontinued. 2009 reactivation (36) of toxoplasmosis. (B) Treatment with artemiside or artemisone resulted in increased survival following reactivation. ARTEMISININ ANALOGUES INHIBIT TOXOPLASMA . with 10 mg/kg) every day for 8 days. 4A). and artemisone. and imaged every second day. Rather. 2014 by guest FIG. Numbers are not available for control mice. have been shown to be more effective against T. Although these two compounds were able to control the initial phases of reactivated infection. which occurs during the first few weeks (Fig. Data shown here are combined results from three experiments. gondii and administration of artemisinin derivates. Experiments were repeated four times. (C) Chronic infection in mice as monitored by cyst formation in the brains of CD1 mice. In comparison. Combined results from the three experiments. CD1 mice were infected with 106 tachyzoites of type II (PRULuc-GFP) parasites. gondii by artemisinin derivatives. and survival was monitored. and compounds were injected (s. Because IFN-␥⫺/⫺ mice cannot control proliferation of tachyzoites. Graph shows one representative experiment. Two days after discontinuation of the drug. it requires nearmicromolar levels to inhibit in vitro growth by 50% (EC50 is 0. Artemiside. gondii. and hence. all of the control mice died within 10 days (Fig. artemisinin derivatives tested here demonstrate that artemisone and artemiside are the most potent analogs to date in Downloaded from http://aac. such as atovaquone. and 2 days later sulfadiazine (400 mg/kg) was administered for 3 weeks in the drinking water. We com- 4453 FIG. they rapidly succumb to infection following removal of sulfadiazine. Survival of mice was monitored for 25 days. while treatment with artemisone prolonged survival in 60% of mice (Fig. they likely do not act on bradyzoites directly. Luc values represent relative light output.c. 4B). Data shown are combined results from three individual experiments. they did not result in a complete cure. while artemisinin-treated and control mice died.asm. 4. 10 mg/kg). the average luciferase (Luc) value dropped after this point. (A) Murine model of reactivated toxoplasmosis. Artemiside. Numbers represent an estimate of the total number of cysts per brain. compounds were administered for 8 days (s. 32). Following the removal of sulfadiazine.and artemisone-treated mice developed lower cyst numbers in the brain compared to artemisinin-treated mice. n ⫽ 3 to 5 animals per group. mice were challenged with parasites and treated with sulfadiazine to suppress acute infection and allow development of tissue cysts. mice began dying at day 18. n ⫽ 3 or 4 animals per group. This result indicates that artemisinin compounds were not able to eradicate the chronic infection.c.and artemisone-treated mice survived the infection. †† indicates all animals succumbed to infection.and artemisonetreated mice were able to control the infection. Inhibition of in vivo growth of T.org/ on April 22. † indicates 4 of 5 animals succumbed to infection. CD1 mice were infected i. (A) Survival of mice following oral challenge with T. which has previously been tested using a similar model (10. 3. Several artemisinin derivatives. are shown. At day 0. with 106 tachyzoites of type II (PRU-Luc-GFP) T. DISCUSSION Previous studies have shown that although artemisinin is somewhat active against T.) every day for 8 days. Treatment was then removed to allow reactivation. artesunate. thus. Similar outcomes are seen with treatment with other drugs that are nonetheless capable of controlling replication of tachyzoites. In contrast.p. 4B).. gondii in vitro. pared the efficacy of the most potent artemisinin derivatives using this in vivo model. treated with compounds (10 mg/kg s. (B) Parasite burdens in mice during acute infection as measured by luciferase imaging.8 ␮M) (27). 53. Inhibition of reactivation of chronic toxoplasmosis by artemisinin derivatives.VOL. IFN-␥⫺/⫺ mice were infected orally with 20 cysts of PRU-LucGFP. and all mice eventually died by day 25 (data not shown). which occurs by a process of stage conversion from bradyzoite to tachyzoite. The majority of the artemiside. which succumbed to infection (†).

Both of these compounds were effective in reducing mortality during acute challenge in the mouse model. exhibit at least threefold greater activity against both chloroquine-sensitive and -resistant strains of P. Treatment with pyrimethamine and sulfadiazine (with folic acid supplementation) is recommended in cases of confirmed fetal infection. Here. A previous study using artemether. such as artemiside. Our studies do not suggest that either compound acts directly on bradyzoites within tissue cysts. remains to be established. Moreover. Development of less fetotoxic derivatives of artemisinin may be of use for preventing congenital infections. where more lipophilic compounds. To be effective in treating reactivation of chronic toxoplasmosis. Rather. 8 uM for artemisinin [17] versus 0. Artemisone also possesses negligible neurotoxicity both in vitro and in vivo (28). ANTIMICROB. Artemisone displayed greater efficacy than artesunate (approximately threefold) in phase II trials with nonsevere-malaria patients (19). and the failure to observe antitoxoplasmal activity may relate to characteristics of the rat model. Nonetheless.. The log P values (log10 of the partition coefficient) for selected compounds are given in Fig. gondii. 34). Spiramycin does not cross the placental barrier and so is not effective in treating infection in the fetus. spiramycin is used for such treatment in Europe. The ability to control tachyzoite replication may have utility for treatment of either primary acute toxoplasmosis or reactivation of chronic infection. compounds need to penetrate the blood-brain barrier. owing to a lack of efficacy against bradyzoites within tissue cysts (22). New drugs capable of controlling acute replication would also be valuable for use during pregnancy. although the reasons for this discrepancy are unknown. few studies have been performed to evaluate in vivo efficacies. We have previously found that the more lipophilic aminoartemisinin derivatives tend to be more active as antimalarial drugs in vitro. Hence. At present. Artemisone has been shown to be safe in human phase I trials. Although artemisinin derivatives do not directly address this deficiency. it is possible to suppress active replication of tachyzoites but allow development of tissue cysts (38). the efficacy of artemisinins against more diverse strains of T. when maternal infection may threaten to spread to the developing fetus. 1 and reflect their relative hydrophobicity as previously determined (10). Artemisone is a 10-alkylaminoartemisinin that was developed as a potent antimalarial drug (13). however. this may reflect pharmacokinetic parameters of the murine model (i. Ocular infections are often severe in these regions. This correlation is also seen in vivo. AGENTS CHEMOTHER. the studies demonstrate that artemisone (log P ⫽ 2.8 uM for our studies [27]).49) and artemiside (log P ⫽ 4. thus representing patient populations for which new therapies with reduced toxicity and fewer side effects may be particularly valuable. we have selected compounds with a range of lipophilicities in order to establish how important this is in relation to in vivo activities. gondii and P. gondii in vitro. In contrast.4454 DUNAY ET AL. We also tested both artemisone and artemiside in a murine model for reactivation that simulates the situation in immunocompromised patients.asm. an analogue that has slightly lower activity in vitro. berghei and Plasmodium yoelii (10).97) are the most active of the compounds. They were also slightly better than a series of semisynthetic alkylated derivatives of artemisinin that have been further described elsewhere (13). Despite this improved safety in humans. more effective treatments that have fewer side effects would be beneficial. they may offer some potential for extending the spectrum of therapies available for toxoplasmosis. which account for most human infections (1. the neurotoxicity of the current clinically used derivatives of artemisinins. Other reports comparing artemisinin and artemether and several synthetic analogues report much higher EC50s (i. it is clear from the in vitro data shown in Fig. Ocular toxoplasmosis is generally recognized to result from reactivation of congenital infection. this treatment can be given only after the first trimester (26). We observed that artemiside and artemisone were approximately equipotent and were about eightfold more effective than artemisinin and about twofold more active than artesunate. We tested these derivatives only against the rapidly growing type I strain RH. Although artemisinin and its derivatives are partially effective in inhibiting T. Although current antibiotic therapy is available for toxoplasmosis. and previous studies have shown that it is less neurotoxic and antiangiogenic than DHA (8). Furthermore. Based on their in vitro efficacy and favorable pharmacokinetic and pharmacodynamic properties. although rats are more susceptible to these effects than rabbits (31). However. such as artesunate or DHA. it carries the added risk of adverse drug reactions. however. artemisone has shown fetal toxicity in a rat pregnancy model.. present a concern if protracted dose loadings are to be used against targets other than malaria. and it is conceivable that they might have different potencies against slower-growing type II strains. gondii in vitro and in vivo. gondii in vitro.e. which have recently been recognized as common in regions such as South America (18. This study used relatively high doses of compounds (18 and 100 mg/kg). carinii (6). ocular infections can occur following newly acquired infections in immunocompetent adults (16. there was an accompanying reduction in the chronic burden of tissue cysts in the CNS. yet in some regions. By treating IFN-␥⫺/⫺ mice with sulfadiazine. It demonstrated excellent pharmacodynamics and pharmacokinetics. despite the lack of controlled trials showing efficacy (26).org/ on April 22. notably southern Brazil. 2014 by guest terms of inhibiting growth of T. a host that is highly resistant to toxoplasmosis. it suffers from problems of intolerance to sulfa drugs combined with a requirement for long-term maintenance. more-rapid turnover and/or clearance). we tested artemisone and artemiside using in vivo models for toxoplasmosis. 15). Pyrimethamine-sulfadiazine treatment has been shown to improve outcomes in some cases (23). our studies demonstrate that several artemisinin derivatives show promise for inhibiting T. 2 that there is no simple relationship between lipophilicity and efficacy. Re- Downloaded from http://aac. failed to show efficacy in a rat model of combined infection with T. Although the doses of compounds necessary to achieve this effect are quite high. 20). we chose to explore the activities of several artemisinin derivatives in more susceptible murine models for acute and chronic toxoplasmosis. Rather. including humans. Hence. the effective concentrations used here cannot be directly extrapolated to other hosts. Therefore. and it was well tolerated by subjects in phase I trials (28). the protective outcome is likely due to the overall reduced parasite burden due to inhibition and/or killing of tachyzoites in vivo.e. .

D. M. Petersen. H. C. Med. 2001. B. Downloaded from http://aac. Moreno. L. In D. Sibley. Liesenfeld. Infect. Muller. Croft. Globalization and the population structure of Toxoplasma gondii. Toxoplasmosis: a comprehensive clinical guide. P. L. I. L. Remington. In D. Baatz. P. Agents Chemother. In the absence of IFN-␥. Clin. Dis. D. Montoya. H. and mice were also partially protected from lethal reactivation by treatment with artemisone. Arasteh. G. Remarkable in vitro and in vivo activities of the hydroxynaphthoquinone 566C80 against tachyzoites and tissue cysts of Toxoplasma gondii. and D. Breakthrough cerebral toxoplasmosis in a patient receiving atovaquone prophylaxis after a hematopoietic stem cell transplant. H. Nagamune. G. 29. Leport. M. 2002.). Infect. Chau. S. Chirgwin. L. J. Org. and J. and J.. The corresponding studies for artemiside have not been carried out.D. Stewart. 12:582–587. and such regimens could provide an alternative for treatment in patients that fail to tolerate sulfa drugs. Norfolk. Jiang. was partially supported by a fellowship from the DFG. Recent transcontinental sweep of Toxoplasma gondii driven by a single monomorphic chromosome. Voith. Romer. B. Yolken. C. Joynson.. Q.. 103:162–166. Infect. W. Silveira. Hattenbach. Huskinson. Artemifone.. 53. B. P. Joynson. M. United Kingdom. Curr.. Infect.and 10␤-aryl derivatives of dihydroartemisinin. K. and O. Lack of activity of artemether for prophylaxis and treatment of Toxoplasma gondii and Pneumocystis carinii infections in rat. D. Fitzner. H. Ed. R. R. Antimicrob. M. 22. Yuan. Acad. Fillisetti. Radtke. 2007. Wei. D. Krishna. Noble. Previous studies have used a very similar model based on knockout of the interferon consensus sequence binding protein (ICSBP) to demonstrate the efficacy of atovaquone for controlling reactivation in the mouse model of toxoplasmosis (10. 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