Clindamycin, which has been reported to have no significant in vitro activity against Toxoplasma gondii, actually markedly inhibits the growth of this parasite in infected human fibroblasts. When measured 3 days after treatment, the concentration required to reduce parasite growth by 50%o is about 1 ng/ml. Some observers failed to note this inhibition because of its markedly delayed onset. At 6 ng/ml, clindamycin is parasiticidal, and the rate and extent of parasite killing increase with higher drug concentrations. With the aid of chemical mutagenesis, we isolated a parasite mutant that is approximately 100-fold more resistant to clindamycin than is the wild type. Lincomycin inhibits T. gondii at a higher 50% inhibitory concentration, about 100 ng/ml. The clindamycin-resistant mutant is partially cross-resistant to lincomycin.While Derouin et al. (3) observed that clindamycin concentrations as low as 0.5 ng/ml inhibited the growth of Toxoplasma gondii, at least three other laboratories have reported that the drug has no in vitro activity (5,6,8). These latter observations are in marked contrast both to the well-established efficacy of clindamycin phosphate against toxoplasmosis in infected mice (1, 6) and to the preliminary evidence for the successful use of clindamycin phosphate to treat toxoplasmic encephalitis in AIDS patients (2). We show here that clindamycin is actually a potent parasiticidal agent in cultured cells infected with T. gondii, with a 50% inhibitory concentration (IC50) that is among the lowest reported for any antitoxoplasma drug. This activity previously escaped detection by several laboratories because the drug has little effect on parasite multiplication until many cell divisions have occurred. MATERIALS AND METHODSThe host cells for all experiments were confluent cultures of human fibroblasts that were maintained as previously described (13), except that the medium for growth contained 10% calf serum and that for infection contained 1% fetal bovine serum. The parasites were our cloned line (13) of the RH strain or mutants derived from this clone. The parasites were maintained by serial subculturing in human fibroblasts. Infected cells were disrupted by forced extrusion through a 25-gauge needle to release intracellular parasites. The newly released parasites were assayed by a plaque procedure (13). In all final experiments, this procedure was carried out with 25-cm2 flasks and the plaques were counted without staining. For preliminary plaque assays with multiwell trays, the monolayer was first fixed with 0.3 N trichloroacetic acid and then stained with Coomassie blue. Parasites were cloned by the detection of a single plaque 6 days after infection of 0.15-cm2 wells with suitably diluted suspensions of extracellular T. gondii.

2,4-Diaminopteridines (21 compounds) and 2,4-diamino-5-methyl-5-deazapteridines (34 compounds) along with three 2,4-diamino-5-unsubstituted-5-deazapteridines and four 2,4-diaminoquinazolines, each with an aryl groups attached to the 6-position of the heterocyclic moiety through a two-atom bridge (either CH2NH, CH2N(CH3),CH2S, or CH2CH2), were synthesized and evaluated as inhibitors of the growth of Toxoplasma gondii in culture and as inhibitors of dihydrofolate reductase enzymes from T. gondii, Pneumocystis carinii, and rat liver. Exceptionally high levels of combined potency and selectivity as growth inhibitors of T. gondii and as inhibitors of the microbial enzymes relative to the mammalian enzyme were found among the 5-methyl-5-deazapteridines but not for the other heterocyclic types. Thirty of the 34 5-methyl-5-deaza compounds gave growth inhibition IC50 values lower than that of pyrimethamine (0.4 microM) with 14 compounds below 0.1 microM, values that compare favorably with those for piritrexim and trimetrexate (both near 0.02 microM). As inhibitors of T gondii DHFR, all but three of the 34 5-methyl-5-deaza compounds gave IC50 values in the order of magnitude with those of piritrexim (0.017 microM) and trimetrexate (0.010 microM), and 17 compounds of this group gave IC50 values versus P. carinii DHFR similarly comparable with those of piritrexim (0.031 microM) and trimetrexate (0.042 microM). Thirteen of these congeners gave both T. gondii growth inhibition and DHFR inhibition IC50 values of 0.10 microM or less, thus indicating facile penetration of the cell membrane. Eleven of these inhibitors of both T. gondii growth and DHFR have selectivity ratios (IC50 rat liver divided by IC50 T. gondii) of 5 or greater for the parasite DHFR. The highest selectivity ratio of nearly 100 belongs to the 5-methyl-5-deaza compound whose 6-substituent is CH2CH2C6H3(OCH3)2-2,5. This compound is over 10(3)-fold more selective for T. gondii DHFR than bridge homologue piritrexim (selectivity ratio 0.088), a compound now in clinical trials. The candidate with CH2NHC6H3(CH3)2-2,5 in the 6-position gave the highest P. carinii DHFR selectivity ratio of 4.0, which is about 60-fold more selective than trimetrexate (0.071) and 80-fold more selective than piritrexim (0.048) toward this enzyme. The 10 best compounds with respect to potency and selectivity includes six compounds bearing 2,5-disubstituted phenyl groups in the side chain (with little, if any, difference in effects of methyl, methoxy, or ethoxy), two side chains bearing 1-naphthyl groups, and two with 5,6,7,8-tetrahydro-1-naphthyl groups. Bridge groups represented in the 10 choice compounds are CH2NH, CH2N(CH3), CH2CH2, and CH2S. The high levels of both potency and selectivity among these agents suggest that in vivo studies now underway may lead to agents that could replace trimetrexate and piritrexim in treatment of toxoplasmosis and P. carinii pneumonia.

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Azithromycin and spiramycin markedly inhibited the growth of Toxoplasma gondii in cultured human fibroblasts. However, 3 days of treatment were required to reveal their full antitoxoplasma activity. This delayed onset of inhibition was similar to that previously reported for clindamycin. Mutants of T. gondii resistant to azithromycin (AziR_l) and spiramycin (SprR-1) were isolated and compared with a previously described mutant resistant to clindamycin (ClnR-2). Mutant ClnR-2 was cross-resistant to all three antibiotics, while AziR_1 was cross-resistant only to spiramycin and SprR-1 was cross-resistant only to azithromycin. In short-term studies of protein synthesis by freshly prepared extracellular parasites, clindamycin and azithromycin were effective only at concentrations much greater than their 50% inhibitory concentrations in infected cultures and the resistant mutants did not differ from the wild type in antibiotic sensitivity. Thus, protein synthesis on cytoplasmic ribosomes of the parasite did not seem to be the target of these antibiotics. To determine whether mitochondrial protein synthesis in T. gondii was inhibited by clindamycin or azithromycin, wild-type parasites were grown in cultured cells in the presence of antibiotic concentrations well above the 50%o inhibitory concentrations. Mitochondrial function, measured by oxygen uptake per purified extracellular parasite, did not decrease substantially, after the parasites had multiplied 11-fold in the presence of antibiotic.Thus, mitochondrial protein synthesis did not seem to be the target of clindamycin or azithromycin. An alternative target is protein synthesis in the putative apicomplexan organelle that has a 35-kb genome.Toxoplasmic encephalitis is common among AIDS patients. The standard treatment of this opportunistic infection with pyrimethamine and sulfadiazine is generally effective. However, since many AIDS patients cannot tolerate prolonged administration of these drugs (11), alternative treatments are needed. Among the antibiotics known to be active against Toxoplasma gondii are the lincosamide clindamycin and the macrolides spiramycin and azithromycin. Clindamycin reduces mortality in infected mice (1,12,18) and, in combination with pyrimethamine, is as effective as pyrimethamine-sulfadiazine in the treatment of toxoplasmic encephalitis in AIDS patients (7,14). Spiramycin is active against murine toxoplasmosis (2) and is thought to reduce the incidence and severity of congenital disease when used to treat women who acquired toxoplasmosis during pregnancy (17). Azithromycin reduces mortality in experimental murine toxoplasmosis (2). The mechanism of action of these antibiotics against T. gondii is unknown. An understanding of how they exert their antiparasitic activity might allow the design of more potent derivatives. One way to approach the mechanism of action is to characterize antibiotic-resistant mutants. We have isolated a mutant of T. gondii that is markedly resistant to clindamycin (21) and describe here mutants selected for resis...