Lindsay D S, Butler J M, Rippey N S, Blagburn B L
Department of Pathobiology, Auburn University, AL 36849-5519, USA.
Am J Vet Res. 1996 Jan;57(1):68-72.
To examine the efficacies of combinations of 7 sulfonamides and 5 dihydrofolate reductase/thymidylate synthase (DHFR/TS) inhibitors against tachyzoites of Neospora caninum in cultured cells. Mutant tachyzoites that were resistant to pyrimethamine were produced and examined for resistance to other DHFR/TS inhibitors.
After 5 days of treatment, a cell culture flask lesion-based assay was used to determine efficacies of combinations of sulfonamides and DHFR/TS inhibitors against N caninum tachyzoites and to evaluate the sensitivity of pyrimethamine-resistant mutants of N caninum to test agents. Cultured cells that were infected with the appropriate strains of N caninum and treated or not treated (controls) with test agents were examined. Mutations were induced by chemical mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine or by selection for growth in permissive concentration of pyrimethamine.
Synergism was detected for combinations of pyrimethamine, ormetoprim, trimethoprim, or diaveridine with the sulfonamides. Methotrexate did not have improved efficacy when combined with sulfonamides. Two mutants were produced that were resistant to pyrimethamine. Both mutants were resistant to other DHFR/TS inhibitors. Both mutants remained resistant to pyrimethamine in the absence of continuous exposure to the agent, indicating that the induced resistance was stable. Synergism was detected for combinations of DHFR/TS inhibitors and sulfonamides against these pyrimethamine-resistant mutants.
Combinations of suboptimal concentrations of sulfonamides with suboptimal concentrations of DHFR/TS inhibitors results in improved efficacy of the agents in a cell culture assay. Stable resistance to pyrimethamine can be induced in N caninum tachyzoites by use of chemical mutagenesis or by selection.
In vitro evidence indicated that combination treatment, using sulfonamides and DHFR/TS inhibitors, may be effective in treating neosporosis.
研究7种磺胺类药物与5种二氢叶酸还原酶/胸苷酸合成酶（DHFR/TS）抑制剂联合使用对培养细胞中犬新孢子虫速殖子的疗效。制备对乙胺嘧啶耐药的突变速殖子，并检测其对其他DHFR/TS抑制剂的耐药性。
处理5天后，采用基于细胞培养瓶损伤的试验来确定磺胺类药物与DHFR/TS抑制剂联合使用对犬新孢子虫速殖子的疗效，并评估犬新孢子虫乙胺嘧啶耐药突变体对受试药物的敏感性。检查感染了适当犬新孢子虫菌株并接受或未接受（对照）受试药物处理的培养细胞。通过用N-甲基-N'-硝基-N-亚硝基胍进行化学诱变或通过在允许浓度的乙胺嘧啶中选择生长来诱导突变。
检测到乙胺嘧啶、奥美普明、甲氧苄啶或二甲氧苄啶与磺胺类药物联合使用具有协同作用。甲氨蝶呤与磺胺类药物联合使用时疗效并未提高。制备了2个对乙胺嘧啶耐药的突变体。这两个突变体均对其他DHFR/TS抑制剂耐药。在没有持续接触该药物的情况下，这两个突变体对乙胺嘧啶仍保持耐药性，表明诱导的耐药性是稳定的。检测到DHFR/TS抑制剂与磺胺类药物联合使用对这些乙胺嘧啶耐药突变体具有协同作用。
在细胞培养试验中，次优浓度的磺胺类药物与次优浓度的DHFR/TS抑制剂联合使用可提高药物疗效。通过化学诱变或选择可在犬新孢子虫速殖子中诱导出对乙胺嘧啶的稳定耐药性。
体外证据表明，使用磺胺类药物和DHFR/TS抑制剂进行联合治疗可能对治疗新孢子虫病有效。
