Compound 566C80, 2-[trans-4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone, was studied for its in vitro and in vivo activities against Toxoplasma gondii. Replication within human foreskin fibroblasts of tachyzoites of seven different strains, five of them isolated from AIDS patients, was inhibited by concentrations as low as 4.8 x 10-9 M. In vivo, a dose of 100 mg/kg of body weight per day, administered by gavage for 10 days, protected 100% of mice against death due to infection with five different strains of T. gondii, including the highly virulent RH strain. A dose of 50 mg/kg/day protected at least 80% 'of mice infected with the same inoculum, and a dose as low as 9.3 mg/kg/day protected 40 to 60% of mice. Treatment with 50 mg/kg/day for 30 days completely eradicated parasites from mice infected with four of five strains of T. gondii. 566C80 was active in vitro against the cyst stage of T. gondii at concentrations of 50 to 100 ,ug/ml. In vivo activity against this form of T. gondii was examined in mice infected for 6 weeks with strain ME49 and then treated orally with 100 mg of 566C80 per kg per day for 8 weeks. Treated mice sacrfficed at 2-week intervals revealed a steady decline in the numbers of cysts in their brains compared with untreated controls. In addition, mortality as well as clinical signs of brain infection was absent from treated mice, whereas control mice had a high mortality rate and showed clinical signs of central nervous system infection. These results reveal remarkable in vitro and in vivo activities of 566C80 against T. gondii.

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The in vivo activities of three bisphosphonates were determined against Leishmania donovani and Toxoplasma gondii. Alendronate was essentially inactive against both parasites. Pamidronate was active against L. donovani by intravenous administration. Risedronate had a 50% effective dosage of five 2.6-mg/kg of body weight intraperitoneal doses against L. donovani-infected mice but was less effective against T. gondii-infected mice.

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An ELISA for IgA toxoplasma antibodies was positive in 12 pregnant women who seroconverted during gestation. Positive IgA titers were also noted in 10 individuals with biopsy-proven toxoplasmic lymphadenitis; the highest titers were noted in the first months following onset of clinical signs. Toxoplasma IgA antibodies were also demonstrable in 8 of 9 infants/fetuses with congenital toxoplasma infection. In some, IgM antibodies could not be demonstrated. Among 20 patients with AIDS and biopsy-proven toxoplasmic encephalitis, only 1 had IgA antibodies. None of 20 individuals with chronic toxoplasma infection had demonstrable IgA antibodies. Demonstration of IgA toxoplasma antibodies should be useful for diagnosis of recently acquired infection and for diagnosis of the infection in the fetus and newborn.

We have attempted to define the serologic criteria for diagnosis of toxoplasmosis in heart transplant recipients. Of 31 patients who were seronegative before transplantation, 4 received a heart from a seropositive donor, and 3 of these 4 had seroconversion and developed life-threatening toxoplasmosis; the remaining 27 did not have seroconversion or develop clinical toxoplasmosis. Of 19 patients who had antibodies to Toxoplasma before transplantation, 10 developed significant increases in test titers of the dye test or double-sandwich IgM enzyme-linked immunosorbent assay but did not develop a clinical illness that could be attributed to toxoplasma infection. Significant serologic changes occurred more often in patients who received azathioprine, corticosteroids, and antithymocyte globulin than in those who received cyclosporine, corticosteroids, and antithymocyte globulin (p less than 0.05). These data show the wide clinical spectrum and differences in kinetics of antibody response of patients who develop toxoplasma infection after transplantation, and suggest that clinical disease occurs in those who have seroconversion but is rare in patients with preexisting antibody who have serologic evidence of recrudescence.

We have investigated the activity of 60 bisphosphonates against the replication of Toxoplasma gondii in vitro and of three of the most active compounds, in vivo. The two most active compounds found were n-alkyl bisphosphonates containing long (n = 9 or 10) hydrocarbon chains, not the nitrogen-containing species used in bone resorption therapy. The target of all of the most active bisphosphonates appears to be the isoprene biosynthesis pathway enzyme farnesyl pyrophosphate synthase (FPPS), as indicated by the correlations between T. gondii growth inhibition and FPPS (human and Leishmania major) enzyme inhibition and by the fact that a T. gondii strain engineered to overexpress FPPS required considerably higher levels of bisphosphonates to achieve 50% growth inhibition, while the IC(50) for atovaquone (which does not inhibit FPPS) remained the same in the overexpressing strain. The phosphonate inhibitor of the non-mevalonate pathway, fosmidomycin, which inhibits the enzyme 1-deoxyxylulose-5-phosphate reductoisomerase, had no effect on T. gondii growth. To investigate structure-activity relationships (SARs) in more detail, we used two three-dimensional quantitative SAR methods: comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), to investigate all 60 bisphosphonates. Both the CoMFA and CoMSIA models indicated a 60-70% contribution from steric interactions and a 30-40% contribution from electrostatic interactions and using four N = 55 training sets for each method, we found on average between a factor of 2 and 3 error in IC(50) prediction. The three most active compounds found in vitro were tested in vivo in a Smith-Webster mouse model and the two most active bisphosphonates were found to provide up to an 80% protection from death, a considerable improvement over that found previously with nitrogen-containing bisphosphonates. This effect may originate in the much higher therapeutic indices of these alkyl bisphosphonates, as deduced from in vitro assays using LD(50) values for growth inhibition of a human cell line. Overall, these results indicate that alkyl bisphosphonates are promising compounds for further development as agents against Toxoplasma gondii growth, in vivo.

The cyst form of Toxoplasma gondii has been implicated as a cause of recrudescence of the latent infection in congenitally infected patients and in the immunocompromised host. A method was developed to evaluate the effect of drugs on the cyst form of the parasite and used to evaluate a variety of therapeutic agents. The most active compounds against the cyst form in vitro were arprinocid-N-oxide, azithromycin, and the hydroxynaphthoquinone 566C80.