Antiproliferative Effects and Mechanism of Action of SCH 56592 against Trypanosoma (Schizotrypanum) cruzi: In Vitro and In Vivo Studies

We have investigated the antiproliferative effects of SCH 56592, a new experimental triazole, against Trypanosoma (Schizotrypanum) cruzi, the etiological agent of Chagas’ disease in Latin America. SCH 56592 blocked the proliferation of the epimastigote form of the parasite in vitro at 30 nM, a concentration 30- to 100-fold lower than that required with the reference compounds ketoconazole and itraconazole. At that concentration all the parasite’s endogenous sterols (ergosterol, 24-ethyl-cholesta-5,7,22-trien-3β-ol, and its 22-dihydro analogs), were replaced by methylated sterols (lanosterol and 24-methylene-dihydrolanosterol), as revealed by high-resolution gas chromatography coupled with mass spectrometry. This indicated that the primary mechanism of action of the drug was inhibition of the parasite’s sterol C-14α demethylase. Against the clinically relevant intracellular amastigote form, grown in cultured Vero cells at 37°C, the MIC of SCH 56592 was 0.3 nM, again 33- to 100-fold lower than that of ketoconazole or itraconazole. In a murine model of acute Chagas’ disease, SCH 56592 given at ≥ 10 mg/kg of body weight/day for a total of 43 doses allowed 85 to 100% survival and 90 to 100% cure of the surviving animals, as verified by parasitological, serological, and PCR-based tests, while ketoconazole given at 30 mg/kg day allowed 60% survival but only 20% cure. In a murine model of chronic Chagas’ disease, SCH 56592 was again more effective than ketoconazole, providing 75 to 85% protection from death, with 60 to 75% parasitological cures of the surviving animals, while no parasitological cures were observed with ketoconazole. The results indicate that SCH 56592 is the most powerful sterol biosynthesis inhibitor ever tested against T. cruzi and may be useful in the treatment of human Chagas’ disease.

本研究考察了新型实验性三唑类化合物SCH 56592对克鲁斯锥虫（Trypanosoma (Schizotrypanum) cruzi）的抗增殖活性，该寄生虫是拉丁美洲恰加斯病（Chagas’ disease）的病原体。SCH 56592在30 nM浓度下即可在体外阻断该寄生虫上鞭毛体（epimastigote）的增殖，该浓度比参比药物酮康唑（ketoconazole）和伊曲康唑（itraconazole）所需浓度低30~100倍。通过高分辨气相色谱-质谱联用法（high-resolution gas chromatography coupled with mass spectrometry）检测发现，在此浓度下，寄生虫的所有内源性甾醇（麦角固醇（ergosterol）、24-乙基-胆甾-5,7,22-三烯-3β-醇（24-ethyl-cholesta-5,7,22-trien-3β-ol）及其22-二氢类似物（22-dihydro analogs））均被甲基化甾醇（methylated sterols，羊毛甾醇（lanosterol）和24-亚甲基二氢羊毛甾醇（24-methylene-dihydrolanosterol））替代，这表明该药物的主要作用机制是抑制寄生虫的甾醇C-14α去甲基化酶（sterol C-14α demethylase）。针对临床相关的、在37℃培养的Vero细胞（Vero cells）中增殖的细胞内无鞭毛体（amastigote），SCH 56592的最低抑菌浓度（MIC）为0.3 nM，同样比酮康唑或伊曲康唑低33~100倍。在急性恰加斯病（acute Chagas’ disease）小鼠模型中，以≥10 mg/kg体重/日的剂量给予SCH 56592，共给药43次后，受试动物的存活率可达85%~100%，存活动物的治愈率为90%~100%，该结果经寄生虫学检测（parasitological tests）、血清学检测（serological tests）及基于PCR的检测（PCR-based tests）验证；而以30 mg/kg/日的剂量给予酮康唑时，动物存活率仅为60%，治愈率仅为20%。在慢性恰加斯病（chronic Chagas’ disease）小鼠模型中，SCH 56592的疗效仍优于酮康唑：可使75%~85%的动物免于死亡，存活动物的寄生虫学治愈率为60%~75%，而酮康唑组未观察到任何寄生虫学治愈情况。本研究结果表明，SCH 56592是目前针对克鲁斯锥虫测试过的活性最强的甾醇生物合成抑制剂（sterol biosynthesis inhibitor），有望用于人类恰加斯病的治疗。