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The following metabolites of sulfadiazine (S) were isolated from monkey urine by preparative HPLC: 5-hydroxysulfadiazine (5OH), 4-hydroxysulfadiazine (4OH) and the glucuronide (5OHgluc) and sulfate conjugate of 5OH (5OHsulf). The compounds were identified by NMR, mass and infrared spectrometry and hydrolysis by beta-glucuronidase. The analysis of S, the hydroxymetabolites (4OH, 5OH) and conjugates N4-acetylsulfadiazine (N4), 5OHgluc and 5OHsulf in human and monkey plasma and urine samples was performed using reversed-phase gradient HPLC with UV detection. In plasma, S and N4 could be detected in high concentrations, whereas the other metabolites were present in only minute concentrations. In urine, S, the metabolites and conjugates were present. The limit of quantification of the compounds in plasma varies between 0.2 and 0.6 microgram/ml (S 0.31, N4 0.40, 4OH 0.20, 5OH 0.37, 5OHgluc 0.33 and 5OHsulf 0.57 microgram/ml). In urine it varies between 0.6 and 1.1 micrograms/ml (S 0.75, N4 0.80, 4OH 0.60, 5OH 0.80, 5OHgluc 0.80 and 5OHsulf 1.1 micrograms/ml). The method was applied to studies with healthy human subjects and Rhesus monkeys. The metabolites 5OH, 5OHgluc and 5OHsulf were present in Rhesus monkey and not in man. Preliminary results of studies of metabolism and pharmacokinetics in Rhesus monkey and man are presented.

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.