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Sulfadiazine and the metabolites N4-acetyl-sulfadiazine, 4-OH-sulfadiazine, 5-OH-sulfadiazine, 5-OH-glucuronide-sulfadiazine, and 5-OH-sulfate-sulfadiazine were tested separately and in combination with pyrimethamine for the inhibitory activity on Toxoplasma gondii growth in vitro. Except for N4-acetyl-sulfadiazine, all sulfa compounds possessed anti-Toxoplasma activity. The addition of 0.05 micrograms of pyrimethamine per ml, a concentration which in itself is not inhibitory, potentiated the microbial activity of sulfadiazine and its metabolites 100 fold.

The efficacy of the combination of pyrimethamine and sulfadiazine for the treatment of congenital Toxoplasma gondii infection in rhesus monkeys was studied. The dosage regimen for pyrimethamine and sulfadiazine was established by pharmacokinetic studies in two monkeys. Those studies showed that the distributions of both drugs followed a one-compartment model. The serum elimination half-lives were found to be 5.2 h for sulfadiazine and 44.4 h for pyrimethamine. Sulfadiazine reached a maximum concentration in serum of 58.7 micrograms/ml, whereas a maximum concentration in serum of 0.22 micrograms/ml was found for pyrimethamine. Ten monkeys were infected intravenously with T. gondii at day 90 of pregnancy, which is comparable to the second trimester of organogenetic development in humans. Treatment was administered to six monkeys, in whose fetuses infection was diagnosed antenatally. From the moment that fetal infection was proven, the monkeys were treated throughout pregnancy with 1 mg of pyrimethamine per kg of body weight per day and 50 mg of sulfadiazine per kg of body weight per day orally. The therapy was supplemented with 3.5 mg of folinic acid once a week. No toxic side effects were found with this drug regimen. The parasite was no longer detectable in the next consecutive amniotic fluid sample, taken 10 to 13 days after treatment was started. Furthermore, T. gondii was also not found in the neonate at birth. The parasite was still present at birth in three of four untreated fetuses that served as controls. Both drugs crossed the placenta very well. Concentrations in fetal serum varied from 0.05 to 0.14 micrograms/ml for pyrimethamine and from 1.0 to 5.4 micrograms/ml for sulfadiazine. In addition, pyrimethamine was found to accumulate in the brain tissue, with concentrations being three to four times higher than the corresponding concentrations in serum. Thirty percent of the sulfadiazine was found to reach the brain tissue when compared with the corresponding serum concentration. when administered early after the onset of infection, the combination of pyrimethamine and sulfadiazine was clearly effective in reducing the number of parasites in the fetus to undetectable levels.

The effectiveness of spiramycin for the treatment of rhesus monkey fetuses congenitally infected with Toxoplasma gondii was studied. Eight monkeys were infected at day 90 of pregnancy. This is comparable to the second trimester of organogenetic development in humans. Transmission of infection was found prenatally in five of the eight monkeys by detection of the parasite in the amniotic fluid. Treatment with spiramycin (20 mg/kg/day in two intermittent doses given intravenously) was started as soon as fetal infection was proven and was continued until birth. Nine to 14 days after initiation of treatment, the parasite was still detectable in amniotic fluid samples from four of these five cases. However, the parasite was detected only by PCR and not by mouse inoculation. T. gondii was also detected only by PCR in the placenta of one monkey that delivered prematurely. This monkey received spiramycin treatment for only 2 weeks. In the four monkeys that received treatment for about 7 weeks, the parasite was not present at birth in the placenta nor in amniotic fluid or neonatal organs. Spiramycin accumulates mainly in maternal tissues. Although concentrations in neonatal tissue were found to be 5 to 28 times higher than the corresponding concentrations in neonatal serum, the concentrations in neonatal tissue were still 11 to 16 times lower than those found in the mothers. However, no spiramycin was found in the fetal brains. Early treatment with spiramycin may prevent transmission of infection to the fetus but most probably cannot interrupt an existing brain infection, which is the most severe outcome of congenital toxoplasmosis in humans.

Transplacental transfer of spiramycin was investigated in a rhesus monkey model to study whether the antibiotic reaches therapeutic levels in the fetus. Spiramycin concentrations were measured by bioassay and high-performance liquid chromatography. Pharmacokinetic parameters were determined for bioactive spiramycin as measured by the bioassay. Pharmacokinetic pilot studies showed that spiramycin distribution follows a two-compartment model in rhesus monkeys. Following a single intravenous dose of 50 or 250 mg, dose-dependent kinetics were observed. At a dose of 50 mg, 10% of the dose was excreted unchanged in the urine. At the higher dose of 250 mg, an oliguric effect was observed. Spiramycin concentrations in fetal serum were measured over time while the maternal concentration was maintained at a constant level. During a 5-h experiment, a maximum fetal-maternal serum ratio of 0.27 was found. In three fetuses, concentrations in serum and tissue were measured following intravenous administration of 50 mg of spiramycin twice daily to the mother for at least 7 days. The fetal-maternal serum ratios were found to be 0.4 to 0.58 after intravenous administration of the final dose of 50 mg to the mother. It appeared that spiramycin accumulated in the soft tissues, especially in the liver and spleen, of both the mother and the fetus. The concentration in placental tissue appeared to be 10 to 20 times that of the concentration in fetal serum. The concentration of spiramycin in amniotic fluid was about five times higher than the concentration in fetal serum. Another important observation was that absolutely no spiramycin was found in the brain.