naphthoquinones and Toxoplasmosis--Animal

Establishment of a mouse model for congenital toxoplasmosis based on oral infection with oocysts from Toxoplasma gondii ME49 and its application for investigating chemotherapeutic options against congenital toxoplasmosis.. CD1 mice were mated, orally infected with 5, 25, 100, 500 or 2000 oocysts and monitored for clinical signs and survival of dams and pups until 4 weeks post partum . The parasite burden in infected mice was quantified by real-time PCR in lungs, brains and, in the case of surviving pups, also in eyes. Seroconversion was assessed by ELISA. T. gondii cysts in brain were identified by immunofluorescence. In a second experiment, pregnant CD1 mice challenged with 20 oocysts/mouse were treated with buparvaquone or the calcium-dependent protein kinase 1 inhibitor bumped kinase inhibitor (BKI)-1294 and the outcome of infection was analysed.. T. gondii DNA was detected in the brain of all infected animals, irrespective of the infection dose. Seroconversion occurred at 3 weeks post-infection. Most pups born to infected dams died within 1 week post partum , but a small fraction survived until the end of the experiment. T. gondii DNA was detected in the brain of all survivors and half of them exhibited ocular infection. Chemotherapy with both compounds led to dramatically increased numbers of surviving pups and reduced cerebral infection. Most efficient were treatments with BKI-1294, with 100% survivors and only 7% brain-positive pups.. BKI-1294 and buparvaquone exert excellent activities against transplacental transmission in pregnant mice.

Topics: Animals; Antiprotozoal Agents; Disease Models, Animal; Female; Infectious Disease Transmission, Vertical; Male; Mice; Naphthalenes; Naphthoquinones; Piperidines; Pyrazoles; Toxoplasmosis, Animal; Toxoplasmosis, Congenital; Treatment Outcome

Toxoplasmosis is a widely disseminated disease caused by Toxoplasma gondii, an intracellular protozoan parasite. Standard treatment causes many side effects, such as depletion of bone marrow cells, skin rashes and gastrointestinal implications. Therefore, it is necessary to find chemotherapeutic alternatives for the treatment of this disease. It was shown that a naphthoquinone derivative compound is active against T. gondii, RH strain, with an IC50 around 2.5 μM. Here, three different naphthoquinone derivative compounds with activity against leukemia cells and breast carcinoma cell were tested against T. gondii (RH strain) infected LLC-MK2 cell line. All the compounds were able to inhibit parasite growth in vitro, but one of them showed an IC50 activity below 1 μM after 48 h of treatment. The compounds showed low toxicity to the host cell. In addition, these compounds were able to induce tachyzoite-bradyzoite conversion confirmed by morphological changes, Dolichus biflorus lectin cyst wall labeling and characterization of amylopectin granules in the parasites by electron microscopy analysis using the Thierry technique. Furthermore, the compounds induced alterations on the ultrastructure of the parasite. Taken together, our results point to the naphthoquinone derivative (LQB 151) as a potential compound for the development of new drugs for the treatment of toxoplasmosis.

Topics: Animals; Antiprotozoal Agents; Cell Line; Cell Survival; Macaca mulatta; Microscopy, Electron; Naphthoquinones; Structure-Activity Relationship; Toxoplasma; Toxoplasmosis, Animal

The efficacy of three amino-terpenyl naphthoquinones and the alkaloid liriodenine were examined against tachyzoites and tissues cysts of the RH and EGS strains, respectively. Monolayers of 2C4 fibroblasts infected with tachyzoites of the RH strain were incubated with different concentrations of the compounds for 48 h. Specifically, 7-(4-methyl-3-pentenyl)-2-pyrrolidine-[1,4]-naphthoquinone (QUI-5), 6-(4-methyl-3-pentenyl)-2-pyrrolidine-[1,4]-naphthoquinone (QUI-6), 6-(4-methylpentyl)-2-pyrrolidine-[1,4]-naphthoquinone (QUI-11), and 8 h-benzo[g]-1,3-benzodioxolo[6,5,4-de]quinolin-8-one,9Cl-1,2-methylene dioxiaporfina (liriodenine) inhibited intracellular replication of T. gondii. The IC(50) values obtained for compounds QUI-5 and QUI-6 were 69.35 and 172.81 μM (i.e., 21.4 and 53.4 μg/mL), respectively. The naphthoquinone QUI-11 and liriodenine significantly inhibited intracellular replication of T. gondii. The IC(50) values obtained with these experiments were 0.32 and 0.07 μM (i.e., 0.1 and 0.02 μg/mL), respectively. Compounds QUI-5, QUI-6, QUI-11 and liriodenine demonstrated lower toxicity for 2C4 fibroblasts compared to atovaquone. In addition, cysts isolated from the brains of mice chronically infected with the EGS strain were exposed to the compounds. Infectivity of the cysts after incubation with the compounds was assessed by infection of mice. The data obtained showed that in vitro incubation with QUI-6, QUI-11 and liriodenine inhibited the infectivity of the bradyzoites. This activity was time- and concentration-dependent.

Topics: Animals; Antimalarials; Aporphines; Atovaquone; Cells, Cultured; Coccidiostats; Female; Fibroblasts; Foreskin; Humans; Inhibitory Concentration 50; Male; Mice; Naphthoquinones; Structure-Activity Relationship; Sulfadiazine; Toxoplasma; Toxoplasmosis, Animal

The compound 2-hydroxy-3-(1'-propen-3-phenyl)-1,4-naphthoquinone (PHNQ6) was evaluated for activity against Toxoplasma gondii, alone or combined with sulfadiazine. Treatment with PHNQ6 combined with sulfadiazine protected at least 70 and 90% of mice infected with RH and EGS strains, respectively. Mice were treated with PHNQ6 (50 mg/kg/day) alone or combined with sulfadiazine (40 mg/L) 30 days after infection with P strain. The number of brain cysts was lower in mice treated with PHNQ6 alone or combined with sulfadiazine compared to that in control mice. Degenerated bradyzoites were observed in animals treated with PHNQ6. Infectivity of bradyzoites treated with PHNQ6 alone or combined with sulfadiazine was inhibited after in vitro incubation.

Topics: Animals; Antiprotozoal Agents; Drug Therapy, Combination; Female; Mice; Naphthoquinones; Random Allocation; Sulfadiazine; Toxoplasma; Toxoplasmosis, Animal; Treatment Outcome

The efficacy of atovaquone and sulfadiazine was examined alone or in combination for the treatment of mice infected with six Brazilian Toxoplasma gondii strains previously genotyped using the PCR-RFLP assays of the SAG2 gene, in addition to RH strain. Swiss mice were infected intraperitoneally with 10(2) tachyzoites from each strain of T. gondii and treated with 6.25, 12.5, 25 and 50 mg/Kg/day of atovaquone or 40, 80, 160 and 320 mg/Kg/day of sulfadiazine. In a second experiment, mice were treated with the association of previously determined doses of each drug. Treatment started 48 hours post-infection, and lasted 10 days. The susceptibility of T. gondii to atovaquone and to sulfadiazine was different according to the parasite strain. It was observed strains that are susceptible to atovaquone, and strains that are resistant to it. Type I strains were more susceptible to the activity of sulfadiazine and more resistant to atovaquone. Yet type III strains were susceptible to atovaquone and to sulfadiazine. Association of atovaquone and sulfadiazine presented a synergic effect in the treatment of mice infected with RH type I strain and an additive effect in the treatment of mice infected with one type I strain and with two type III strains.

Topics: Animals; Antiprotozoal Agents; Atovaquone; Coccidiostats; Dose-Response Relationship, Drug; Drug Combinations; Drug Synergism; Female; Genotype; Mice; Naphthoquinones; Random Allocation; Sulfadiazine; Toxoplasma; Toxoplasmosis, Animal; Treatment Outcome

Topics: Animals; Antioxidants; Antiprotozoal Agents; Atovaquone; Drug Synergism; Drug Therapy, Combination; Female; Mice; Naphthoquinones; Pyrrolidines; Survival Rate; Thiocarbamates; Toxoplasmosis, Animal

The effect of 14 natural and synthetic naphthoquinones in the replication of Toxoplasma gondii was evaluated. In vitro studies were accomplished in cultures of 2C4 fibroblasts infected with RH-strain. Enzyme-linked immunosorbent assay was used to quantify parasite growth. For the studies in vivo, mice were infected with tachyzoites of the RH strain or cysts of the T. gondii EGS strain. In vitro, seven naphthoquinones demonstrated significant inhibition of intracellular T. gondii growth at concentrations of 1 and 5 micrograms/ml. Only three compounds were significantly protective when tested in animals: 2-hydroxy-3'-(3'-pentenyl)-1,4-naphthoquinone (PHNQ4), 2-hydroxy-3-(1'-vinylphenyl)-1,4-naphthoquinone (PHNQ5), and 2-hydroxy-3-(1'-propen-3'-phenyl)-1,4-naphthoquinone (PHNQ6). In animals infected with the EGS strain and treated with PHNQ4 (50 mg/kg/day orally), a 7-day prolongation of the time to death was observed. Treatment with 100 mg/kg/day orally or 50 mg/kg/day i.p. of PHNQ5 resulted in a 5-day and 16-day prolongation of the time to death, respectively. Treatment with 50 mg/kg/day orally or 50 mg/kg/day i.p. of PHNQ6 resulted in a 4-day prolongation of the time to death or up to 30 days after treatment, respectively. Our results suggest that the naphthoquinones may be important therapeutic agents for the treatment of toxoplasmosis.

Topics: Animals; Antiprotozoal Agents; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Fibroblasts; Humans; Mice; Naphthoquinones; Toxoplasma; Toxoplasmosis, Animal

The efficacy of atovaquone (ATO) combined with clindamycin (CLI) against Toxoplasma gondii was examined in murine models of infection with a mouse-non-virulent (Me49) strain. Swiss-Webster mice inoculated by mouth with 10 or 20 cysts were treated with ATO and CLI alone or combined at dosages of ATO 5-100 and CLI 25-400 mg/kg/day for 2-4 weeks. Drug treatment was initiated (i) day 4 post-infection (acute infection), (ii) 3 months post-infection (chronic infection) and (iii) following a 2-3 week course of treatment with dexamethasone (DXM) alone or combined with cortisone-acetate (CA) introduced 3 months post-infection (reactivated toxoplasmosis). In acute infection, whereas treatment with any drug or drug combination significantly enhanced survival and reduced the brain cyst burden, in mice treated with ATO alone or combined with CLI, the cyst counts were significantly lower than in mice treated with CLI alone. In chronic infection, the decrease in the cyst burden observed 2 weeks after treatment with either drug alone was significant only in mice treated with the combined drugs. Most importantly, in reactivated toxoplasmosis, whereas an effect for the combined drugs was shown in mice suppressed with both DXM alone and combined with CA, in mice pre-treated with DXM a 3 week course of ATO > or = 25 and CLI 50 mg/kg/day significantly increased survival and markedly decreased the cyst burden. The latter effect was long-term, since the cyst burdens in treated mice continued to decrease up to 3 months later, whereas they increased in the untreated mice. The results warrant clinical evaluation of the combination of ATO and CLI in the treatment of toxoplasmosis in both immunocompetent and, more importantly, immunosuppressed patients.

Topics: Acute Disease; Animals; Atovaquone; Brain; Clindamycin; Cysts; Disease Models, Animal; Drug Therapy, Combination; Female; Mice; Naphthoquinones; Toxoplasma; Toxoplasmosis, Animal

Developmental switching in Toxoplasma gondii, from the virulent tachyzoite to the relatively quiescent bradyzoite stage, is responsible for disease propagation and reactivation. We have generated tachyzoite to bradyzoite differentiation (Tbd-) mutants in T. gondii and used these in combination with a cDNA microarray to identify developmental pathways in bradyzoite formation. Four independently generated Tbd- mutants were analysed and had defects in bradyzoite development in response to multiple bradyzoite-inducing conditions, a stable phenotype after in vivo passages and a markedly reduced brain cyst burden in a murine model of chronic infection. Transcriptional profiles of mutant and wild-type parasites, growing under bradyzoite conditions, revealed a hierarchy of developmentally regulated genes, including many bradyzoite-induced genes whose transcripts were reduced in all mutants. A set of non-developmentally regulated genes whose transcripts were less abundant in Tbd- mutants were also identified. These may represent genes that mediate downstream effects and/or whose expression is dependent on the same transcription factors as the bradyzoite-induced set. Using these data, we have generated a model of transcription regulation during bradyzoite development in T. gondii. Our approach shows the utility of this system as a model to study developmental biology in single-celled eukaryotes including protozoa and fungi.

Topics: Animals; Antiprotozoal Agents; Atovaquone; Cell Differentiation; Gene Expression Profiling; Gene Expression Regulation, Developmental; Genes, Protozoan; Genes, Reporter; Green Fluorescent Proteins; Luminescent Proteins; Mice; Mutagenesis; Naphthoquinones; Oligonucleotide Array Sequence Analysis; Phenotype; Protozoan Proteins; Recombinant Fusion Proteins; Selection, Genetic; Toxoplasma; Toxoplasmosis, Animal; Transcription, Genetic; Transcriptional Activation

Immunocompromised patients are at risk of developing toxoplasma encephalitis (TE). Standard therapy regimens (including sulfadiazine plus pyrimethamine) are hampered by severe side effects. While atovaquone has potent in vitro activity against Toxoplasma gondii, it is poorly absorbed after oral administration and shows poor therapeutic efficacy against TE. To overcome the low absorption of atovaquone, we prepared atovaquone nanosuspensions (ANSs) for intravenous (i.v.) administration. At concentrations higher than 1.0 microg/ml, ANS did not exert cytotoxicity and was as effective as free atovaquone (i.e., atovaquone suspended in medium) against T. gondii in freshly isolated peritoneal macrophages. In a new murine model of TE that closely mimics reactivated toxoplasmosis in immunocompromised hosts, using mice with a targeted mutation in the gene encoding the interferon consensus sequence binding protein, i.v.-administered ANS doses of 10.0 mg/kg of body weight protected the animals against development of TE and death. Atovaquone was detectable in the sera, brains, livers, and lungs of mice by high-performance liquid chromatography. Development of TE and mortality in mice treated with 1.0- or 0.1-mg/kg i.v. doses of ANS did not differ from that in mice treated orally with 100 mg of atovaquone/kg. In conclusion, i.v. ANSs may prove to be an effective treatment alternative for patients with TE.

Topics: Animals; Antiprotozoal Agents; Atovaquone; Brain; Chromatography, High Pressure Liquid; Injections, Intravenous; Macrophages, Peritoneal; Male; Mice; Naphthoquinones; Sulfadiazine; Suspensions; Tissue Distribution; Toxoplasma; Toxoplasmosis, Animal

Mice were infected intraperitoneally with 10,000 tachyzoites of Toxoplasma gondii (RH) strain and, 24 h later, were treated orally for 10 days with atovaquone and azithromycin, either alone or in combination. Evaluation of the efficacy of the drugs was performed by microscopic examination of smears prepared from the organs of the mice, and by subinoculation of visceral and brain suspensions from surviving mice into healthy mice at the end of the experiments. It was found that 58%, 83% and 100% of the mice survived after administration of 75, 150 or 200 mg/kg per day of azithromycin, respectively. Moreover, 8%, 17% and 25% of the mice survived after treatment with atovaquone at 20, 50 or 100 mg/kg per day, respectively. No synergistic or additive effects of combinations of atovaquone and azithromycin were observed. However, azithromycin did not eradicate the parasite from the brain and viscera of the infected mice, whereas atovaquone at 20, 50 and 100 mg/kg per day removed the parasite from viscera and at 100 mg/kg per day eradicated the parasite from the brain of infected mice. The combinations of atovaquone and azithromycin failed to completely eradicate the parasite from the brain and viscera of infected mice.

Topics: Animals; Anti-Bacterial Agents; Antiprotozoal Agents; Atovaquone; Azithromycin; Disease Models, Animal; Drug Synergism; Drug Therapy, Combination; Female; Mice; Naphthoquinones; Random Allocation; Toxoplasma; Toxoplasmosis, Animal

The effect of clindamycin (CLI) combined with autovaquone (ATO) was examined in a murine model of acute toxoplasmosis. Swiss Webster mice intraperitoneally infected with 10(2) or 10(4) tachyzoites of the RH strain of Toxoplasma gondii were perorally treated with either drug alone (for ATO, 5, 25, 50, or 100 mg/kg of body weight/day; for CLI, 25, 50, or 400 mg/kg/day) or both combined (for ATO plus CLI, respectively, 5 plus 25, 25 plus 25, 25 plus 50, 50 plus 50, or 100 plus 400 mg/kg/day) starting with day 1 for 14 days. Survival was monitored during 7 weeks. Residual infection was assessed by a bioassay of representative 4-week survivors and by parasite DNA detection by PCR for representative 7-week survivors. An effect of treatment was shown in all treatment groups compared to untreated control mice (P = 0.0000). Among mice infected with 10(2) parasites, ATO and CLI at any dose combination protected significantly more animals than ATO alone (P = 0.0000), but compared to CLI alone, given its good effect, the combined drugs were no more effective (P > 0.05). For mice infected with 10(4) parasites, the drugs combined at the lowest and highest doses (5 plus 25 and 100 plus 400 mg/kg/day) were, similarly, more effective than ATO alone (P = 0.035 and 0.000, respectively) but not than CLI alone (P > 0. 05). However, treatment with ATO plus CLI at 25 plus 25, 25 plus 50, and 50 plus 50 mg/kg/day protected 20, 33, and 78% of mice, respectively, compared to virtually no survivals among those treated with either drug alone (P < 0.0005), thus demonstrating a significant synergistic effect of ATO and CLI against T. gondii. Furthermore, the dose of ATO at a given dose of CLI was shown to be critical to the effect. Moreover, the absence of residual infection in some survivors shows the potential of this drug combination to eliminate the parasite.

Topics: Acute Disease; Animals; Anti-Bacterial Agents; Antiprotozoal Agents; Atovaquone; Brain; Clindamycin; Dose-Response Relationship, Drug; Drug Synergism; Female; Lung; Mice; Naphthoquinones; Polymerase Chain Reaction; Toxoplasma; Toxoplasmosis, Animal

Two 3-alkyl-substituted 2-hydroxy-1,4-naphthoquinones, NSC 113452 (NSC52) and NSC 113455 (NSC55), were evaluated for activity against Toxoplasma gondii in vitro and in murine models of acute toxoplasmosis. In vitro, both NSC52 and NSC55 significantly inhibited intracellular replication of T. gondii. In vivo, each compound was examined alone and in combination with other drugs currently used for treatment of human toxoplasmosis. Although none of the compounds protected mice against death when administered orally, both were significantly protective when administered intraperitoneally. In addition, a significant increase in survival was observed when suboptimal doses of each compound were used in combination with suboptimal doses of pyrimethamine or sulfadiazine. These results indicate that combinations of NSC52 or NSC55 with pyrimethamine or sulfadiazine have promising activity against T. gondii.

Topics: Animals; Antiprotozoal Agents; Cells, Cultured; Dose-Response Relationship, Drug; Drug Therapy, Combination; Female; Humans; Mice; Naphthoquinones; Toxoplasma; Toxoplasmosis, Animal

The aim of this work was to develop a new pharmaceutical form of atovaquone and to study its activity against Toxoplasma gondii in vitro and in vivo. Nanocapsules were chosen as the oral dosage form of administration. An analytical method was developed to determine the drug content in nanocapsules. The stability of these nanocapsules were assessed by following drug content, size, pH and osmolarity for a period of six months. The in vitro activity of atovaquone-loaded nanocapsules against tachyzoites of T. gondii (RH stain) was comparable to its suspension form. In vivo studies were carried out in murine models of acute and chronic toxoplasmosis. Mice acutely infected with the virulent RH strain were orally treated with a dose regimen of 15 mg/kg/day for 10 days, starting from day 1 post-infection. 75% of the mice receiving atovaquone-loaded nanocapsules survived 30 days post-infection, compared to none of untreated controls and none of mice treated with the suspension with the same dose regimen. In mice chronically infected by the COUL or the ME49 strain (Type II strains), then treated for six weeks, treatment with atovaquone (15 mg/kg/d, nanoparticles or suspension) resulted in a decrease of brain parasitic burden, which was significantly more pronounced in ME49-infected mice and in those treated with drug-loaded nanocapsules. These results show that the sensibility of T. gondii to atovaquone is different according to the strains and that the activity of atovaquone in the treatment of toxoplasmosis is enhanced when administered in nanoparticular form.

Topics: Acute Disease; Administration, Oral; Animals; Antiprotozoal Agents; Atovaquone; Brain; Capsules; Chronic Disease; Colloids; Disease Models, Animal; Drug Carriers; Drug Stability; Female; Lung; Mice; Naphthoquinones; Parasitemia; Solubility; Survival Rate; Suspensions; Temperature; Toxoplasma; Toxoplasmosis, Animal

Because combination therapy is required to treat human toxoplasmosis, we examined combinations of the ketolides HMR 3004 and HMR 3647 with atovaquone, clindamycin or sulphadiazine in a murine model of toxoplasmosis. An oral dose of 50 mg/kg/day of HMR 3004 protected 30% of mice lethally infected with Toxoplasma gondii. The same dose protected 100% of infected mice when administered in combination with non-protective doses of atovaquone, clindamycin or sulphadiazine. Similar results were noted with 25 mg/kg/day of HMR 3647. These results demonstrate that these drug combinations are highly effective for treating toxoplasmosis in mice.

Topics: Animals; Anti-Bacterial Agents; Antiprotozoal Agents; Atovaquone; Clindamycin; Drug Therapy, Combination; Female; Ketolides; Macrolides; Mice; Naphthoquinones; Sulfadiazine; Toxoplasma; Toxoplasmosis, Animal

The capacity of interleukin 12 (IL-12) to potentiate drugs in the treatment of murine toxoplasmosis was examined. IL-12 (100 ng/injection), atovaquone (10 mg/kg of body weight/day), or clindamycin (5 mg/kg/day) administered alone caused delayed time to death or minimal survival rates. In contrast, significant survival rates resulted when the same dose of IL-12 was used in combination the same doses of atovaquone (P=0.01) or clindamycin (P=0.001). Infected mice treated with IL-12 plus drug produced significantly higher levels of gamma interferon than controls. Although IL-12 was effective only when administered before infection, these results suggest that this cytokine may be a useful adjunct in the therapy of human toxoplasmosis in situations when cysts reactivate and tachyzoites start multiplying in immunocompromised patients.

Topics: Animals; Anti-Bacterial Agents; Antiprotozoal Agents; Atovaquone; Clindamycin; Drug Synergism; Female; Interferon-gamma; Interleukin-12; Interleukin-2; Interleukin-4; Mice; Mice, Inbred BALB C; Naphthoquinones; Toxoplasmosis, Animal

Current therapy for toxoplasmosis with a synergistic combination of pyrimethamine plus sulfadiazine or pyrimethamine plus clindamycin is not always efficacious and is frequently discontinued due to intolerable toxic effects in immunocompromised individuals, particularly those with AIDS. Trovafloxacin, a new fluoroquinolone with potent activity against Toxoplasma gondii, was examined for potential synergistic activity when combined with other drugs used for treatment of human toxoplasmosis. Combinations of trovafloxacin with clarithromycin, pyrimethamine, or sulfadiazine demonstrated significantly enhanced activities compared to those observed with each drug alone. Our results suggest that combinations of trovafloxacin and other anti-toxoplasma drugs should be further explored for treatment of toxoplasmosis in humans.

Topics: Acute Disease; Administration, Oral; Animals; Anti-Infective Agents; Antiprotozoal Agents; Area Under Curve; Atovaquone; Clarithromycin; Drug Synergism; Drug Therapy, Combination; Female; Fluoroquinolones; Mice; Naphthoquinones; Naphthyridines; Rifabutin; Tissue Distribution; Toxoplasmosis, Animal

The efficacy of rifabutin (RIFA) alone or in combination with atovaquone (ATO) was examined in vitro and in a murine model of acute toxoplasmosis. In vitro studies were performed with MRC5 fibroblast tissue cultures, with quantification of Toxoplasma growth by enzyme-linked immunosorbent assay. For in vivo studies, mice were acutely infected with 10(4) tachyzoites of the virulent RH strain and were then treated perorally for 10 days from day 1 or day 4 postinfection. The efficacy of each drug regimen was assessed by determination of survival rates and sequential titration of parasites in blood, brain, and lungs by a tissue culture method. In vitro, RIFA was inhibitory for Toxoplasma growth at concentrations between 0.5 and 20 micrograms/ml; the 50% inhibitory concentration was estimated to be 1.68 micrograms/ml. When RIFA and ATO were combined, synergistic effects were noted for RIFA at 20 micrograms/ml combined with ATO at 0.01 or 0.02 microgram/ml and RIFA at 1, 2, or 5 micrograms/ml combined with ATO at 0.02 microgram/ml. In vivo, administration of RIFA at 200 mg/kg of body weight per day from day 1 to day 10 resulted in a 100% protection during treatment, with clearance of parasites from the blood, brain, and lungs. After the cessation of therapy, relapses occurred in the brain and lungs; the mortality was 46% at the end of the experiment (day 30). Among the mice treated with RIFA at 200 mg/kg/day from day 4 to day 14, no death was recorded during the treatment period and a marked reduction in parasite burdens was observed in blood and tissues; however, relapses occurred and 10% of mice survived until day 30. Administration of RIFA at 200 mg/kg/day in combination with ATO at 100 mg/kg/day resulted in a marked prolongation of survival compared with that for mice that received ATO or RIFA alone. However, in mice receiving the combination, parasite burdens in blood and organs were similar to those in mice treated with RIFA alone. These results confirmed the activity of RIFA in the treatment of acute toxoplasmosis and the potential of the combination of RIFA-ATO since the two drugs act synergistically against Toxoplasma gondii.

Topics: Animals; Anti-Bacterial Agents; Antiprotozoal Agents; Atovaquone; Brain; Drug Synergism; Enzyme-Linked Immunosorbent Assay; Female; Mice; Naphthoquinones; Rifabutin; Survival Analysis; Toxoplasma; Toxoplasmosis, Animal

Prophylactic efficacy of antimicrobial agents against pneumocystosis and toxoplasmosis was examined in a model of concurrent Pneumocystis carinii and Toxoplasma gondii infections in rats. Corticosteroid-treated rats naturally infected by P. carinii were challenged with the RH strain of T. gondii. Infection was assessed by counting P. carinii cysts in lung and by titration of T. gondii in tissues by tissue culture. Untreated rats died after challenge, with P. carinii infection in lungs and T. gondii infection in liver, spleen, lungs, and brain. In rats that received trimethoprim-sulfamethoxazole or pyrimethamine plus dapsone, T. gondii was eradicated and P. carinii pneumonia prevented. Roxithromycin, 200 or 400 mg/kg, provided significant protection against toxoplasmosis but had no efficacy against P. carinii. Atovaquone, 100 or 200 mg/kg, had only partial efficacy against pneumocystosis and toxoplasmosis. These results definitively confirm use of trimethoprim-sulfamethoxazole and pyrimethamine plus dapsone for prophylaxis against combined infection in immunocompromised hosts.

Topics: Animals; Antifungal Agents; Antiprotozoal Agents; Atovaquone; Brain; Dapsone; Drug Therapy, Combination; Liver; Lung; Male; Mice; Naphthoquinones; Pneumocystis; Pneumocystis Infections; Pyrimethamine; Rats; Rats, Wistar; Roxithromycin; Spleen; Toxoplasma; Toxoplasmosis, Animal; Trimethoprim, Sulfamethoxazole Drug Combination

The morphological effects of drug treatment with atovaquone in the brains of mice chronically infected with Toxoplasma gondii was examined by light and electron microscopy. As early as 1 and 2 weeks of treatment there appeared to be fewer tissue cysts compared to untreated controls and this reduction was more significant after 4 weeks treatment. There also appeared to be a decrease in the number of inflammatory nodules and the severity of the meningitis. Ultrastructurally, the cysts of both treated and control animals were located within host cells. There was a marked increase in both the number of cysts with lysed bradyzoites and the number of degenerate bradyzoites after 4 weeks treatment. It is probable that the drug is more active against the metabolically active immature bradyzoites than the mature organisms. Drug treatment does not appear to result in rupture of tissue cysts or release of Toxoplasma antigens since there is a reduction rather than an increase in the inflammatory response. This drug may be useful in treating chronic toxoplasmosis since it appears to be active against the bradyzoites reducing the parasite burden (cyst number) without initiating a destructive inflammatory response.

Topics: Animals; Antiprotozoal Agents; Atovaquone; Brain; Chronic Disease; Female; Mice; Mice, Inbred CBA; Microscopy, Electron; Naphthoquinones; Toxoplasmosis, Animal; Toxoplasmosis, Cerebral

Rifabutin, a semisynthetic derivative of rifamycin S, was examined alone and in combination with other drugs for activity in treatment of systemic toxoplasmosis and toxoplasmic encephalitis in murine models. One hundred percent of the mice infected with a lethal inoculum of tachyzoites or cysts of Toxoplasma gondii were protected against death by treatment with doses of 400 or 300 mg of rifabutin per kg administered alone for 10 days. Doses of 200 mg/kg protected at least 80% of the mice, and doses of 100 mg/kg protected 10 to 40% of the infected mice against death. Doses of 50 mg/kg were not protective but caused a delay in time to death. Combination of nonprotective (50-mg/kg) or slightly protective (100-mg/kg) doses of rifabutin with doses of sulfadiazine, pyrimethamine, clindamycin, or atovaquone that did not confer any protection against death from toxoplasmosis when administered alone resulted in remarkable enhancement of the in vivo activities of all of these drugs. Seventy-five percent of the infected mice survived when treated with 100 mg of rifabutin per kg per day combined with the ineffective dose of 10 mg of pyrimethamine per kg. A dose of 50 mg of rifabutin per kg in combination with the ineffective dosages of clindamycin (25 mg/kg/day), atovoquone (5 mg/kg/day), and sulfadiazine (80 mg per liter of drinking water) protected at least 80, 60, and 60% of the mice against death, respectively. The inflammatory responses in the brains of mice treated for 30 days with 200 mg of rifabutin per kg per day were significantly reduced compared with those in the brains of untreated controls. These observations suggest that clinical trials with rifabutin for treatment and prevention of human toxoplasmosis may be justified, particularly when the drug is used in combination with other drugs with activity against T. gondii.

Topics: Animals; Antiprotozoal Agents; Atovaquone; Brain; Clindamycin; Drug Therapy, Combination; Female; Mice; Mice, Inbred CBA; Naphthoquinones; Pyrimethamine; Rifabutin; Sulfadiazine; Toxoplasmosis, Animal; Toxoplasmosis, Cerebral

The efficacy of atovaquone alone or combined with pyrimethamine, sulfadiazine, clarithromycin, and minocycline was examined in vitro and in a murine model of acute toxoplasmosis. In vitro studies were performed with MRC5 fibroblast tissue cultures, with quantification of Toxoplasma growth by an enzyme-linked immunosorbent assay. For in vivo studies, mice were acutely infected intraperitoneally with 10(4) tachyzoites of the virulent RH strain and then treated perorally for 10 days from day 1 postinfection. The following drug regimens were investigated: atovaquone at 100 and 50 mg/kg of body weight per day and the combinations of atovaquone at 50 mg/kg with sulfadiazine at 200 mg/kg, pyrimethamine at 12.5 mg/kg, clarithromycin at 200 mg/kg, or minocycline at 50 mg/kg. Efficacy was assessed by determination of survival rates and sequential determination of parasite burdens in blood, brain, and lungs. In vitro, atovaquone inhibited Toxoplasma growth at a concentration of > or = 0.02 mg/liter; the 50% inhibitory concentration was estimated to be 0.023 mg/liter. No synergistic effect was observed when it was combined with sulfadiazine, clarithromycin, or minocycline, whereas a significant antagonistic effect was noted for the combination of atovaquone with pyrimethamine. In vivo, administration of atovaquone at 100 or 50 mg/kg/day for 10 days resulted in prolonged survival compared with that in untreated mice; this survival was associated with a reduction of parasite burdens in blood and tissues during the course of treatment. The combinations of atovaquone with pyrimethamine, clarithromycin, or sulfadiazine were more efficient than each drug administered alone, in terms of survival, but parasite burdens in blood and organs were not reduced compared with those in mice treated with any of the agents alone. These experimental results confirmed the activity of atovaquone against Toxoplasma gondii, but no marked improvement in efficacy was observed in vitro and in vivo when this drug was combined with pyrimethamine, sulfadiazine, minocycline, or clarithromycin.

Topics: Animals; Antiprotozoal Agents; Atovaquone; Clarithromycin; Coccidiostats; Drug Combinations; Drug Interactions; Enzyme-Linked Immunosorbent Assay; Kinetics; Mice; Minocycline; Naphthoquinones; Pyrimethamine; Sulfadiazine; Toxoplasma; Toxoplasmosis, Animal

The activity of atovaquone in the treatment of murine toxoplasmosis was greatly enhanced when administered in combination with pyrimethamine or sulfadiazine. Mice infected with lethal inocula of tachyzoites or cysts of Toxoplasma gondii and treated with doses of atovaquone, pyrimethamine, or sulfadiazine that were ineffective when administered alone had 70% survival when pyrimethamine plus atovaquone and 100% survival when sulfadiazine plus atovaquone was used. Of interest, doses of pyrimethamine and, particularly, sulfadiazine far below the doses that would induce any protection in infected mice were active when combined with atovaquone. These results suggest that clinical trials for treatment of toxoplasmosis in AIDS patients using the combination of atovaquone with sulfadiazine or pyrimethamine are justified.

Topics: Animals; Antiprotozoal Agents; Atovaquone; Drug Synergism; Drug Therapy, Combination; Female; Mice; Naphthoquinones; Pyrimethamine; Sulfadiazine; Toxoplasmosis, Animal

Toxoplasmosis is a frequent opportunistic infection in patients with AIDS. In these patients the major immune deficiencies are a severe depletion of CD4+ T lymphocytes and an impaired capacity to produce IFN-gamma. A mouse model was developed and used to study the effects that depletion of CD4+ T cells and/or inhibition of the protective activity of IFN-gamma have on the effectiveness of the drug therapy for toxoplasmosis. Infection of mice with a lethal inoculum of Toxoplasma gondii cysts followed by treatment with the hydroxynaphthoquinone 566C80 or with sulfadiazine resulted in 100% survival whereas untreated controls had 100% mortality within 15 days of infection. Administration of antiserum to IFN-gamma resulted in early death of untreated mice and in 30% mortality in those treated. Administration of mAb to CD4+ T cells followed by infection with T. gondii prevented the development of both antibody and cell-mediated immune responses against the parasite. These mice resisted the acute infection while undergoing specific treatment. Discontinuation of the treatment, however, resulted in reactivation of the infection and the majority of the animals died within 17 days of suspension of the treatment. Administration of antiserum to IFN-gamma or to CD4+ T cells 24 h but not 15 days after conclusion of the treatment also resulted in mortality. These results indicate that successful treatment of toxoplasmosis depends on the status of the immune system, particularly of CD4+ T cells. Although it is speculative to compare results obtained in mice to the situation in humans, our work suggests that restoration of a competent immune response is of crucial importance for a successful treatment of toxoplasmosis in immunocompromised individuals.

Topics: Animals; Antibodies, Protozoan; Antiprotozoal Agents; Atovaquone; CD4-Positive T-Lymphocytes; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Hypersensitivity, Delayed; Immunity, Cellular; Interferon-gamma; Lymphocyte Depletion; Mice; Naphthoquinones; Toxoplasmosis, Animal; Treatment Failure

The in vitro and in vivo activities of the hydroxynaphthoquinone 566C80 against the cyst form of Toxoplasma gondii were evaluated. In vitro treatment (100 micrograms of 566C80 per ml for 3 days) of cysts isolated from brains of mice infected for 1, 2, 3, 4, or 9 months resulted in loss of viability of the cysts and did not reveal any influence of the duration of in vivo infection on sensitivity to the drug. In vivo experiments to determine the effect of prolonged treatment with 200 mg of 566C80 per kg of body weight per day on cysts in brains of CBA/Ca mice infected with strain ME49 revealed a steady and significant decline in the numbers of cysts compared with the numbers in untreated controls. Histopathology of brains from control mice revealed inflammatory infiltrates around capillaries and in the parenchymas and meninges which were consistently less evident in the brains of treated mice. In addition, cysts were rarely observed in treated mice, whereas extensive inflammation and large numbers of cysts were found throughout the entire brain in control mice infected for the same period. The reduction in the numbers of cysts was evident as early as day 5 of treatment but was more marked at 8 weeks of treatment. The numbers of cysts in the brains of Swiss Webster mice infected for 3 or 6 months also significantly decreased following treatment for 15 or 30 days with the same dose of 566C80. Our results indicate that 566C80 has excellent activity against cysts of T. gondii both in vivo and in vitro and that sensitivity of the cysts to 566C80 is not affected by the duration of the infection in vivo.

Topics: Animals; Antiprotozoal Agents; Atovaquone; Brain; Female; Mice; Mice, Inbred CBA; Naphthoquinones; Toxoplasmosis, Animal

Novel hydroxynaphthoquinones are reported with outstanding efficacy against Plasmodium, Eimeria and Theileria species. Biochemical evidence is presented for the selective toxicity of these compounds being due to inhibition of parasite respiratory systems.

Topics: Animals; Antiprotozoal Agents; Apicomplexa; Cattle; Chickens; Coccidiosis; Eimeria; Malaria; Mice; Naphthoquinones; Plasmodium berghei; Plasmodium falciparum; Protozoan Infections; Theileriasis; Toxoplasma; Toxoplasmosis, Animal