Abstract
Azoles such as posaconazole (Posa) are highly potent against Trypanosoma cruzi. However, when tested in chronic Chagas disease patients, a high rate of relapse after Posa treatment was observed. It appears that inhibition of T. cruzi cytochrome CYP51, the target of azoles, does not deliver sterile cure in monotherapy. Looking for suitable combination partners of azoles, we have selected a set of inhibitors of sterol and sphingolipid biosynthetic enzymes. A small-scale phenotypic screening was conducted in vitro against the proliferative forms of T. cruzi, extracellular epimastigotes and intracellular amastigotes. Against the intracellular, clinically relevant forms, four out of 15 tested compounds presented higher or equal activity as benznidazole (Bz), with EC50 values ≤2.2 μM. Ro48-8071, an inhibitor of lanosterol synthase (ERG7), and the steroidal alkaloid tomatidine (TH), an inhibitor of C-24 sterol methyltransferase (ERG6), exhibited the highest potency and selectivity indices (SI = 12 and 115, respectively). Both were directed to combinatory assays using fixed-ratio protocols with Posa, Bz, and fexinidazole. The combination of TH with Posa displayed a synergistic profile against amastigotes, with a mean ΣFICI value of 0.2. In vivo assays using an acute mouse model of T. cruzi infection demonstrated lack of antiparasitic activity of TH alone in doses ranging from 0.5 to 5 mg/kg. As observed in vitro, the best combo proportion in vivo was the ratio 3 TH:1 Posa. The combination of Posa at 1.25 mpk plus TH at 3.75 mpk displayed suppression of peak parasitemia of 80% and a survival rate of 60% in the acute infection model, as compared to 20% survival for Posa at 1.25 mpk alone and 40% for Posa at 10 mpk alone. These initial results indicate a potential for the combination of posaconazole with tomatidine against T. cruzi.
Highlights
Chagas disease (CD), a vector-borne anthropozoonosis endemic in the American continent, is caused by the protozoan parasite Trypanosoma cruzi (Chagas, 1909)
As a strategy to overcome the limitation of CYP51 inhibitors, we have proposed to combine them with a partner drug that either acts in the same pathway, sterol biosynthesis, or that inhibits a functionally linked pathway, sphingolipid synthesis (Fügi et al, 2015)
Most of the tested compounds showed quite relevant toxicity towards mammalian host cells, leading to low selectivity indices (SIs), except for Ro48-8071 and tomatidine hydrochloride (TH), which presented promising SIs of 12 and 115, respectively (Table 2). Based on their high activity against intracellular amastigote T. cruzi and good selectivity towards the mammalian host cells, Ro48-8071 and tomatidine were moved to in vitro combination assays with the reference drug for CD (Bz) and two others that displayed efficacy in in vitro and in vivo assays of T. cruzi experimental infection: the imidazole CYP51 inhibitor Posa and the nitroimidazole Fexinidazole (Fexi) (Table 3)
Summary
Chagas disease (CD), a vector-borne anthropozoonosis endemic in the American continent, is caused by the protozoan parasite Trypanosoma cruzi (Chagas, 1909). Oral transmission due to beverages contaminated with the feces or with infected triatomines currently represents a serious challenge in many endemic areas such as Brazil (Coura et al, 2014; Dias, 2017). This neglected disease presents a short acute phase with patent parasitemia, which is usually asymptomatic or oligosymptomatic with “flu-like” symptoms (Prata, 2001; Rassi et al, 2010). After years or even decades, about 30% of the patients in the chronic phase develop progressive cardiac or gastrointestinal injuries (Ribeiro et al, 2012; Malik et al, 2015)
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