Recent advances in drug development for Chagas ...

Salomão K, De Castro SL (2017) Chapter 8: Recent advances in drug development for Chagas disease: two magic words, combination and repositioning. In: Different Aspects on Chemotherapy of Trypanosomatids, Leon L & TorresSantos EC (Eds.), Nova Science Publishers, NY, ISBN 978-1-53610-850-7, pp. 181-226.

Chapter

RECENT ADVANCES IN DRUG DEVELOPMENT FOR CHAGAS DISEASE: TWO MAGIC WORDS, COMBINATION AND REPOSITIONING Kelly Salomão and Solange Lisboa de Castro

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Laboratório de Biologia Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil

ABSTRACT Chagas disease is one of the seventeen neglected tropical diseases, and approximately 8 million people are infected with T. cruzi worldwide. This disease is characterized by an acute phase and a chronic phase, and the aetiologic treatment still depends on benznidazole (Bz) and nifurtimox (Nif), which were empirically developed in the 1970s. The major limitations of these nitrocompounds are their limited and variable curative ability towards the established chronic form and their toxic effects. In the search for alternatives to treat Chagas disease, an increasing number of drug development programmes are ongoing, involving networks of researchers, non-governmental organizations and pharmaceutical companies performing large-scale screenings of compounds using transgenic parasites and resulting in a switch from target-based screening towards whole-parasite-based assays. Several of these studies have focused on C14α-sterol demethylase (CYP51), cruzain inhibitors and nitrocompounds. New series of azolic and non-azolic compound inhibitors of CYP51 and reversible and irreversible inhibitors of cruzain have been submitted for preclinical testing. A long list of 3-nitro-triazole-based derivatives has been investigated; some derivatives display high efficacy in vitro and in vivo and adequate ADMET characteristics, and some derivatives are substrates of a specific T. cruzi nitroreductase type I (TcNTR). The strategies of multitarget drug design, repositioning and combinations of drugs are commonly used strategies today. Although advances have been made in the screening technologies and new knowledge of the biology of T. cruzi has been discovered, in the near future, we envisage two routes to achieve new options for the aetiologic

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Corresponding author: [email protected].

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Kelly Salomão and Solange Lisboa de Castro treatment of Chagas disease: new schemes using Bz or Nif that reduce the doses and durations of treatment and their combination with azoles.

1. CHAGAS DISEASE AND T. CRUZI Chagas disease, caused by Trypanosoma cruzi, was discovered in 1909 by the Brazilian physician Carlos Chagas (1879-1934) (Andrade et al., 2011; Dias, 2015). Its estimated global prevalence was 7 million people by 1960, 16-18 million people by 1990, 9.8 million people by 2006 and 5-7 million people by 2010 (Schofield et al., 2006; PAHO, 2010; WHO, 2015). The life cycle of T. cruzi involves passage through two hosts: briefly, in a mammalian host, intracellular amastigotes and bloodstream trypomastigotes; and in a haematophagous triatomine, epimastigotes. There is a broad variety of parasite subpopulations with differences in virulence, pathogenicity and drug susceptibility. Based on biological and biochemical parameters, the different strains have been organized following different classifications, such as zymodemes (Romanha et al., 1979; Miles & Cibulskis, 1986), schizodemes (Morel et al., 1980) and biodemes (Andrade & Magalhães, 1996). In 1999, an Expert Committee recommended the clustering of the parasite strains into two major groups, called T. cruzi I and T. cruzi II (Anonymous, 1999). Later, the parasite strains were grouped into six discrete typing units (DTUs), named TcI to TcVI (Zingales et al., 2009, 2012; Guhl & Ramirez, 2011), defined as “sets of stocks that are genetically more related to each other than to any other stock and that are identifiable by common genetic, molecular or immunological markers” (Tibayrenc, 1998).

1.1. Globalization Chagas disease, classically associated with poor and rural populations, underwent an urbanization process in the 1970s and 1980s to Latin American cities and later beyond endemic countries. The spread to North America, Europe, Asia and Australia has continually increased, creating new epidemiological, economic, social and political challenges (Schmunis, 2007; Schmunis & Yadon, 2010; Jackson et al., 2014). As of 2013, there were an estimated 36.7 million people who had migrated out of Latin America (Conners et al., 2016). In non-endemic countries, the transmission of Chagas disease is associated with the congenital route, blood transfusions and organ transplantation (Bern et al., 2011; Rodriguez-Guardado et al., 2015). With regard to congenital transmission, seven cases in Spain (Oliveira et al., 2010), two cases in Switzerland (Jackson et al., 2009) and one case in Sweden (Pehrson et al., 1981) have been reported. In the US, applying the seroprevalence number reported by the Pan American Health Organization (PAHO, 2006) and assuming a rate of transmission by infected pregnant women of 1-5%, Bern and Montgomery (2009) estimated 300,000 infected persons and 63-316 congenital T. cruzi infections/year. Also, because the beginning of screening of blood donations in 2007, 797 confirmed seropositive donations have been detected, the majority in California, Florida, and Texas. Assuming a rate of transmission of 5%, Buekens et al. (2008) estimated that 40,000 pregnant women and 2,000 newborns could be infected by T. cruzi in North America. For this reason, the Centers for Disease Control and Prevention (CDC) classified Chagas disease as one of the five neglected parasitic infections in the United States (CDC, 2015). Approximately 3.5 million Latin Americans live in Europe, with an estimated 90,000

Recent Advances in Drug Development for Chagas Disease

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infected individuals, roughly 50,000 of whom living in Spain; the majority were from Ecuador, Argentina and Bolivia (WHO, 2010; Gascon et al., 2010). Analysis of the prevalence of Chagas disease in Latin America migrants revealed an infection rate of 4.2% in Europe through 2004, with a high heterogeneity dependent upon the country of origin (Requena-Méndez et al., 2015), and for the period of 2004-2014, it was concluded that this prevalence was higher than expected in some migrant groups and that studies based on the blood donor screening prevalence underestimated the burden of disease (Conners et al., 2016).

1.2. Transmission Chagas disease is transmitted mainly to humans by triatomine vectors, blood transfusions, oral and congenital transmission and less commonly by direct transmission from T. cruzi reservoirs, ingestion of uncooked meat from infected animals, organ transplantation, and laboratory accidents (Deane et al., 1984; Steindel et al., 2008; Altclas et al., 2008; Dias & Amato-Neto, 2011). The formidable decrease in the prevalence of Chagas disease observed from 1990 to 2010 was essentially the consequence of the launching of transnational programmes in Latin America focused on the elimination of domestic vectors and the screening of blood donors: the Southern Cone Initiative, the Andean Countries’ Initiative, the Central American and Mexico Initiative and the Initiative of the Amazon Countries, all supported by PAHO/WHO (Schofield et al., 2006; Guhl, 2007; Dias, 2009; Moncayo & Silveira, 2009). The estimated prevalence of infection per 100 habitants due to vectorial transmission was highest in Bolivia, Argentina and Paraguay, followed by Ecuador, El Salvador and Guatemala (WHO, 2015). Several publications have emphasized the southern US as an area of autochthonous T. cruzi infection, with a large proportion of at-risk households infested by triatomines (Hotez, 2013; Klotz et al., 2014). Woody and Woody (1955) documented the first autochthonous case in a girl in Texas, and soon afterwards, a second paediatric case was also reported (Greer, 1956). More recently, five new autochthonous cases were identified, indicating a risk of human vectorborne transmission in southeast Texas (Garcia et al., 2016). Blood transfusion as a mechanism of Chagas disease transmission was suggested by Dr. Emmanuel Dias in 1945, and the first cases of infection by this route were reported by Pedreirade-Freitas et al. in 1952. To prevent transfusional transmission, Nussenszweig et al. (1953) proposed the use of gentian violet (GV), and subsequently, its use was recommended in blood banks in endemic regions (WHO, 1984). GV showed no noteworthy side effects in patients transfused with treated blood (Rezende et al., 1965; Moraes-Souza et al., 1988), but its use causes blood to turn a purple colour that might stain the skin and mucosa (Wendel, 1998). Mandatory screening of blood products began in many Latin American countries in 1988, and today, 19 of 21 Latin American countries have achieved 100% screening of donated blood (Schmunis, 1999; Moncayo & Silveira, 2009; WHO, 2015). Oral transmission, which is probably the most frequent mechanism among vectors and wild mammals, acquired relevance because triatomines are attracted to peridomiciliar areas in search of blood sources due to the reductions in food reservoirs from extensive deforestation (Abad-Franch & Monteiro, 2007). Between 2000 and 2010, more than 1,000 cases of acute Chagas disease were diagnosed in Latin America, with 71% associated with the ingestion of

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Kelly Salomão and Solange Lisboa de Castro

contaminated food (Shikanai-Yasuda & Carvalho, 2012), such as wild animal meat, vegetables, and fruit juices and pulps (Ianni & Mady, 2005; Pereira et al., 2009). The Brazilian Amazon basin is the main area where these outbreaks occurred, resulting in the classification of the region as endemic for Chagas disease (Coura et al., 1994, 2002; Valente et al., 2009; Coura & Junqueira, 2015a). In past years, an increasing number of acute and chronic cases have been identified (Pinto et al., 2008; Ferreira et al., 2009; Valente et al., 2009; Coura & Junqueira, 2012, 2015b), with cardiac involvement (Pinto et al., 2001; Albajar-Viñas et al., 2003; Barbosa-Ferreira et al., 2010; Santana et al., 2014) and high mortality rates, compared with cases of transmission via triatomine excreta deposition after biting (Rassi et al., 2010). Congenital transmission has become an important source of new cases of Chagas disease, with the success of the programmes of vector and blood bank control (Gebrekristos & Buekens, 2014; Carlier et al., 2015). The estimated number of new cases of congenital T. cruzi infection is 8,668 cases/year (WHO, 2015). In a systematic review of the literature through 2012, it was estimated that the rates of vertical transmission were 5% and 2.7% in endemic and non-endemic areas, respectively (Howard et al., 2014). In Brazil, in a retrospective analysis of congenital transmission, a prevalence of 2% was confirmed in the Central region, where TcII is the main T. cruzi lineage, whereas in the South region where TcV is predominant, this rate was 5%, similar to frequencies reported in Argentina, Paraguay and Bolivia (Luquetti et al., 2015). During the period of 2004-2009 in Bolivia, the National Congenital Chagas Program found in 42,538 children born to positive mothers when analysed at birth, yielding a positive infection rate of 1.4% for Chagas disease (Alonso-de-Vega et al., 2013). In non-endemic countries, standardization, expansion and reinforcement of control strategy programmes for congenital Chagas disease are needed; Catalunya, Spain, is the only region with systematic monitoring of pregnant women from Latin America (Soriano-Arandes et al., 2016).

1.3. Clinical Aspects Chagas disease results from the establishment of T. cruzi in host tissues, involving an initial acute phase followed by a chronic phase, classified as indeterminate, cardiac, or digestive. The acute phase is characterized by circulating parasites detectable in the bloodstream and is frequently asymptomatic. It is estimated that only 1-2% of all individuals acquiring the infection are recognized as being in the acute phase (WHO, 2002); however meningoencephalitis occasionally occurs, particularly in children younger than 2 years old (Rassi et al., 2012). Without treatment, approximately 5-10% of symptomatic patients die during this phase due to encephalomyelitis or severe cardiac failure and rarely due to sudden death (Prata, 2001). After 2-3 months, the infection enters the chronic phase, and without successful treatment, it is life-long. Approximately two thirds of infected individuals have the indeterminate form of the chronic phase, which is asymptomatic and defined by the presence of T. cruzi antibodies and normal electrocardiographic and radiologic exams (Prata, 2001). Several years or even decades after the initial infection, the other one third of infected individuals, due to an unbalanced inflammatory response and persistent low parasitism, will develop symptomatic chronic disease with cardiac (20-30%) and/or digestive (15-20%) disturbances. Chagasic myocarditis is the most common form of non-ischaemic


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cardiomyopathy worldwide and is the most expressive manifestation of the disease, due to its frequency and severity (Higuchi et al., 2003; Marin-Neto et al., 2007; Rassi et al., 2012; Malik et al., 2015). The digestive form of Chagas disease is observed in roughly 15% of chronically infected patients (Prata, 2001), and it is characterized mainly by chronic inflammation and destruction of parasympathetic neurons, leading to progressive enlargement of the oesophagus or colon (Rezende & Moreira, 1988; Peñaranda-Carrillo et al., 2006; Matsuda et al., 2009). In the case of congenital infection, the clinical characteristics are heterogeneous, ranging from asymptomatic/oligosymptomatic cases (60-90% of infected newborns) to severe cases with meningoencephalitis, myocarditis or respiratory distress syndrome (Bittencourt, 1976; Freilij & Altcheh, 1995; Torrico et al., 2004). The treatment of infected children during the first year of life ensures therapeutic success in almost 100% of cases, with a low risk of side effects (Carlier et al., 2011; Altchech et al., 2011), and it potentially decreases the risk of future congenital transmission (Sosa-Estani et al., 2012). Cases of the reactivation of Chagas disease are most commonly diagnosed by the recrudescence of parasitaemia, and other symptoms include subcutaneous nodules, myocarditis, meningitis, encephalitis, and stroke (Campos et al., 2008; Pinazo et al., 2013; Lattes & Lasala, 2014; Perez et al., 2015). In the cases of transplant patients, most of the reported cases of reactivation were related to heart and kidney transplants (Kun et al., 2009). In patients co-infected with HIV, the CNS is the most commonly affected system, with manifestations of acute meningoencephalitis, followed by the cardiac system, with manifestations of acute myocarditis (Sartori et al., 2007; Cordova et al., 2008; Almeida et al., 2011).

1.4. Benznidazole and Nifurtimox The current aetiological treatment for Chagas disease is restricted to two nitroheterocycles drugs: benznidazole (Bz/LAFEPE and Abarax/ELEA) and nifurtimox (Nif, LAMPIT/Bayer) (Figure 1). The results obtained with these drugs vary according to the phase of Chagas disease, the period of treatment and dose, and the age and geographical origin of the patients (Coura & De Castro, 2002). Both drugs have shown successful results with high parasitological cure rates during the acute phase, but the effectiveness decreases with advancement of the infection; therefore, early detection and intervention are crucial for outcomes (Coura & Borges-Pereira, 2011). Their efficacy and tolerance are inversely correlated with the age of the patient, and its side effects occur more frequent in elderly patients (Viotti et al., 2009). The high incidence of collateral effects, especially for adults, has led to treatment abandonment in several instances (Pinazo et al., 2010; Jackson et al., 2010; Perez-Molina et al., 2012). In contrast, children have a markedly higher tolerance of treatment (WHO, 2002; Dias et al., 2016). A recent study with Nif in US, revealed frequent side mild effects, but in most cases they are managed with dose reduction and/or temporary suspension of medication (Forsyth et al., 2016).

1.4.1. Mechanism of Action of Bz and Nif The precise mechanism of action of Bz and Nif is still controversial, although these drugs have been in clinical use for more than four decades. Nitrocompounds acting as prodrugs must undergo activation involving two classes of nitroreductases (NTRs): an oxygen insensitive class catalysing the two-electron reduction of the nitrogroup (NTR-I) and an oxygen-sensitive

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class catalysing one-electron reduction (NTR-II) (Peterson et al., 1979). In the 1980s, the initial studies of both drugs revealed that their activity involved the formation of free radicals and/or electrophilic metabolites. For Nif, the trypanocidal activity was associated with oxidative stress due to drug transformation to a nitroanion radical by NTR-II, redox cycling with molecular oxygen, and the production of reactive oxygen species (ROS) (DoCampo & Stoppani, 1979; Moreno et al., 1982). For Bz, this oxidative damage was not considered the key mode of action because the detection of corresponding nitroanion radicals occurred only at concentrations much higher than the trypanocidal concentrations. It is likely that the trypanocidal action is due to the covalent bonding of the reduced metabolites of Bz to lipids, DNA and proteins (Polak & Richle 1978; Diaz-de-Toranzo et al., 1988). These distinct mechanisms could be explained by the differences in the lower reduction potential of Bz compared with that of Nif, leading to a lower rate of nitroanion radical formation (DoCampo, 1990).

Figure 1. Drugs for the aetiologic treatment of Chagas disease: (a) benznidazole; and (b) nifurtimox.

More recently, Boiani and colleagues (2010) observed a reduction in low-molecularweight thiols in parasites treated with Nif, whereas neither the generation of ROS nor redox cycling was found at trypanocidal concentrations, arguing against oxidative stress as its mode of action. The research group of Wilkinson has defended that a specific nitroreductase of the parasite (TcNTR), an NTR-I, is involved in the mode of action of nitrocompounds through a twoelectron reduction in the nitro group using NADH as a cofactor (Wilkinson et al., 2008). It was previously demonstrated that this NTR-I, also called “old yellow enzyme” (TcOYE), was involved in the metabolization of nitroheterocycles in the parasite (Kubata et al., 2002). The in vitro reduction by TcNTR leads to a hydroxylamine intermediary, which undergoes different transformations: for Nif, the nitrofuran group is transformed into a highly reactive unsaturated open chain nitrile (Hall et al., 2011), whereas for Bz, a consecutive two-electron reduction leads to a dihydroxy-dihydroimidazole derivative, which can decompose to the toxic metabolite glyoxal, which in turn can react with the parasite DNA (Hall & Wilkinson, 2012). However, a metabolomics analysis of T. cruzi treated with Bz revealed drug binding to low-molecularweight thiols and to protein thiols, whereas low-molecular-weight adducts of glyoxal, the toxic end product of Bz metabolism by TcNTR, were not detected (Trochine et al., 2014).

1.4.2. New avenues of Aetiological Treatment In the 1980s, aetiological treatment for chronic stages of Chagas disease was not recommended, pending more solid evidence of its efficacy (Brener, 1982) and based on the


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belief that autoimmunity was a key factor in myocardial damage during this phase (Kalil & Cunha-Neto, 1996). However, over the last 20 years, the role of T. cruzi in triggering tissue damage was determined, and clinical trials have demonstrated the efficacy of Bz treatment in chronic patients, including those with early cardiomyopathy (Viotti et al., 2014). The group of Viotti in Argentina performed several studies using f low doses of Bz (5 mpk for 30 days) in the treatment of chronic patients. A study with a median follow-up of 10 years showed lower levels of disease progression (4% vs. 14%) and the development of ECG abnormalities (5% vs. 16%) in treated patients, compared with the untreated group (Viotti et al., 2006). A subsequent study, with a follow-up period of 3 years, revealed that treated patients presented significantly decreased levels of T. cruzi antibodies (64%) compared with those of the untreated group (21%), and the seronegative conversion rate was 40% vs. 7% (Viotti et al., 2011). Because the discontinuation of treatment is a primary drawback, several studies have compared chronic patients treated with Bz (5 mpk) in whom treatment was interrupted due to side effects with those who completed the treatment. Pinazo et al. (2013) observed no significant differences in the serum levels of the drug between the two groups, and the range of concentration found (3-6 µg/mL) was previously reported as appropriate for therapeutic doses (Raaflaub, 1980). In another study, 20% of chronic patients submitted to incomplete treatment (Bz 5 mpk, median 10 days) met the criteria for a cure (seronegative conversion on two or three tests (Alvarez et al., 2012). In re-evaluating the optimal schedule of Bz administration, a recent pilot study showed that intermittent Bz (5 mpk) treatment of every 5 days for a total of 60 days led to adverse effects in only 50% of patients, with one case of treatment suspension and a 6% failure rate, indicating a promising therapeutic scheme (Alvarez et al., 2015). Listed below are the recent clinical trials performed with Bz and Nif; in those registered in ClinicalTrials.gov are included in parenthesis the sponsor of the clinical assay, the trial status and principal investigator, as accessed in July, 2016: 

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TRAENA (Etiologic Treatment with Benznidazole in Adult Patients with Chronic Chagas Disease. A Randomized Clinical Trial; NCT02386358) (Instituto Nacional de Parasitologia Dr. Mario Fatala Chaben; completed; Dr. Adelina Rosa Riarte): This phase III, randomized, clinical trial was developed in Argentina to assess whether Bz at 5 mpk for 60 days could inhibit the clinical progression of adult patients with lowrisk, chronic Chagas disease and to compare conventional and nonconventional serology and qPCR as predictors of clinical evolution. After 10 years of follow-up, no significant differences in disease progression were observed between the treated and placebo groups. However, the statistical sampling and the Bz regime used were insufficient to demonstrate the initially estimated clinical impact (Riarte et al., 2016). BENEFIT (BENznidazole Evaluation For Interrupting Trypanosomiasis, NCT00123916 (Population Health Research Institute; completed; Dr. Carlos Morillo): This phase III clinical trial was launched in 2005 in Latin America to clarify the role of therapy with Bz (5 mpk for 40-80 days) in preventing cardiac disease progression in chronic chagasic cardiac patients. The final results were recently published (Morillo et al., 2015), and they concluded that Bz significantly diminished the parasite load circulating in the blood, but this reduction was not correlated with the amelioration of cardiac deterioration. This study also found that clinical progression

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and PCR conversion were significantly more common in patients from Brazil than in those from Colombia and El Salvador (Morillo et al., 2015). CHAGASAZOL (NCT01162967) (Hospital Universitari Vall d'Hebron Research Institute; completed; Dr. Israel Molina): This randomized, open-label, phase II clinical trial performed in Catalunya (Spain) with Bolivian migrants who were chronic chagasic patients without cardiac insufficiency; the patients were administered posaconazole or Bz. After 40 weeks of treatment, treatment failure (positive qPCR) was observed in 80-90% of patients treated with the azole and in 5.9% of those treated with Bz. In all of the treated groups, no serious adverse events were reported (Molina et al., 2014). According to Urbina (2015), treatment failure was related to the suboptimal doses of posaconazole that were used and the administration schedules. Moraes et al. (2014) pointed out that it is important to observe the country of origin of the participants because in Bolivia, DTU V is the most prevalent T. cruzi stock, and in preclinical studies, the azoles have been shown less effective than Bz. E1224 trial (NCT01489228) (DNDi-CH-E1224-001) (DNDi and Esai; completed; Dr. Faustino Torrico): This phase II study was conducted in chronic indeterminate Chagas disease patients in Bolivia using E1224 (prodrug of the azole ravuconazole) and Bz. The parasitological response of all of the patients after a 2-week treatment was sustained for 12 months after treatment in 81% of cases. E1224 proved to be effective in removing the parasite at the end of treatment, without maintaining the elimination of the parasite (Barreira et al., 2016). DNDi-CH-E1224-002 was a phase I study initiated in 2014 in Argentina to evaluate the pharmacokinetic interaction of Bz and E1224 in healthy volunteers, with the objective of planning subsequent studies of drug combinations, and no major safety or tolerability issues were identified (CDCP, 2015). STOPCHAGAS (NCT01377480) (Merck Sharp & Dohme; completed; Dr. Carlos Morillo): This phase II trial conducted in Argentina, Colombia and Mexico evaluated posaconazole, Bz, or their combination in the treatment of patients with indeterminate Chagas disease for 60 days. The percentages of successful response (qPCR) were 10, 15.6, 86.7 and 82.1% for groups treated with placebo, posaconazole, Bz, and posaconazole+Bz, respectively, showing that the azole was not effective as monotherapy (Stopchagas, 2016). CINEBENZ (Population Pharmacokinetics in Benznidazole-treated Adults with Chronic Chagas Disease; NCT01755403) (Barcelona Centre for International Health Research; completed; Dr. Joaquim Gascon): This study was a prospective, open-label, single-centre clinical trial aiming to characterize the pharmacokinetics of Bz in adults with chronic Chagas disease. Dosing simulations showed that a dose of 2.5 mpk adequately maintained Bz plasma concentrations within the recommended target range for the majority of patients (Soy et al., 2015). PEDCHAGAS (Population Pharmacokinetics of Benznidazole in Children with Chagas Disease; NCT00699387) (Hospital de Niños R. Gutierrez de Buenos Aires; completed; Dr. Jaime Altcheh): This study investigated the pharmacokinetics of Bz (5-8 mpk for 60 days) in children receiving treatment for Chagas disease. From the results obtained from the 37 patients who completed the treatment course, it was concluded that Bz was well tolerated, with few mild adverse drug reactions. The Bz


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plasma concentrations were markedly lower than those previously reported in adults (treated with comparable mg/kg doses), possibly due to a higher weight-corrected clearance rate in the small children. The good efficacy of the drug in children, despite the lower levels in blood, raises the possibility that lower Bz doses in adolescents and adults might maintain efficacy while decreasing serious side effects (Altcheh et al., 2014). LACTBENZ (Study of Benznidazole Transfer into Breastmilk in Lactating Women with Chagas Disease, NCT01547533) (Hospital de Niños R. Gutierrez de Buenos Aires; completed; Dr. Facundo Garcia Bournissen): The degree of Bz transfer into the breast milk of lactating women with Chagas disease treated with the drug for 30 days was determined. Of the 12 lactating women enrolled, five had adverse drug events (45%), but no adverse drug reactions or any untoward outcomes were observed in the breastfed infants. The results led Garcia-Bournissen et al. (2015) to conclude that maternal Bz treatment for Chagas disease during breast feeding was unlikely to present a risk to the infant. LACTNFX (Study of Nifurtimox Transfer into Breastmilk in Lactating Women with Chagas Disease, NCT01744405) (Hospital de Niños R. Gutierrez de Buenos Aires; completed; Dr. Facundo Garcia Bournissen): With a similar aim as the clinical trial using Bz, this study examines the transfer of Nif into breastmilk from the blood of lactating women receiving the drug for the treatment of Chagas disease. No study results were posted on ClinicalTrials.gov. Study to Assess Bioequivalence of 30 and 120 mg Nifurtimox Tablets in Chronic Chagas' Patients (NCT01927224) (Bayer; completed): This trial was a phase I, open label, randomized, single dose, cross-over study to assess the bioequivalence between BAY2502 1 x 120 mg, 4 x 30 mg and a slurry of 4 x 30 mg tablets in tap water, following high calorie/high fat meals to adult male and female patients suffering from chronic Chagas disease. The study concluded that in the absence of differences in clinically relevant safety findings, the PK data showed that the 30 mg tablet was a viable formulation for the administration of Nif to children and that the three formulations presented no safety or tolerability issues (Bayer Healthcare, 2015). Study to Assess the Food Effect on the Pharmacokinetics of Nifurtimox Tablets in Chronic Chagas' Patients (NCT02606864) (Bayer; ongoing study): This study will evaluate the effect of food on the absorption of the drug as well as the safety and tolerability of the novel 30 mg tablet (administered as a 120 mg dose) in adults suffering from chronic Chagas disease when administered after a high-fat/high-calorie test meal, compared to a fasting state. BIOMARCHA (New Tools for the Diagnosis, Prognosis and Treatment Follow-up in Chagas Disease; NCT01755377) (Barcelona Centre for International Health Research; ongoing study; Dr. Joaquim Gascón): Biomarkers for prognosis, early diagnosis and the effectiveness of treatment will be investigated. PCR (Optimization of PCR Technique to Assess Parasitological Response for Patients with Chronic Chagas Disease; NCT01678599) (DNDi; information not recently verified): This trial is a single arm, open label study in Bolivia, with all of the enrolled subjects receiving Bz (5 mpk for 60 days).

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Kelly Salomão and Solange Lisboa de Castro 

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CHICO-Bayer (Prospective Study of a Pediatric Nifurtimox Formulation for Chagas' Disease; NCT02625974) (Bayer; ongoing study): This study was designed to examine the effect of Nif in three divided doses in infected children after treatment for 30 days and to evaluate the use of lower doses of this drug as indicated in a previous clinical trials performed with Bz: PEDCHAGAS. CHICAMOCHA 3 - Equivalence of Usual Interventions for Trypanosomiasis (EQUITY); NCT02369978) (Universidad Autónoma de Bucaramanga; ongoing study; Dr. Juan C Villar): This randomized, blinded, parallel-group trial will evaluate the efficacy and safety of Nif and Bz, administered for 60 and 120 days in Colombia and Argentina, to explore regional differences in the treatment effects.

Different strategies are being sought to establish the optimal regimen for Bz in adults with chronic Chagas disease (Viotti et al., 2014; Alvarez et al., 2015). In 2011, Coura and BorgesPereira proposed two lines of conduct for treatment of the chronic phase of Chagas disease: (i) repeated short-term treatments for 30 consecutive days and an interval of 30-60 days for six months to one year; and (ii) combinations of Bz/Nif, Bz or Nif/allopurinol or azoles. Previous studies of chronic patients with Bz (5 mpk) have shown slower clinical progression to cardiomyopathy than in untreated groups (Viotti et al., 1994, 2011), including those receiving incomplete treatment (Alvarez et al., 2012) and cure rates in patients who had to suspend treatment (Alvarez et al., 2012), and a pilot study with the intermittent use of Bz (Alvarez et al., 2015) and the recent CINEBENZ (Soy et al., 2015) and PEDCHAGAS studies (Altcheh et al., 2014) support the hypothesis that a reduced dose of Bz could be used to minimize its toxicity. Different Bz formulations have been investigated, such as formulations with water and polyethylene glycol, to improved Bz solubility (Manarin et al., 2013), as well as extendedrelease tablets (Davanço et al., 2016). The BERENICE (BEnznidazol and triazol REsearch group for Nanomedicine and Innovation on Chagas disease) study, coordinated by Dr. Israel Molina and involving partners in Europe and Latin America, includes the study of novel Bz lipid-based drug delivery systems to improve the pharmacokinetic and pharmacodynamic features of Bz (Berenice, 2016). In this context, the BENDITA (BEnznidazole New Doses Improved Treatment and Associations) study by DNDi is arriving in due time. This study will evaluate new schemes for Bz treatment that reduce dosages in monotherapies and in combination with E1224, and it will be launched in Bolivia, Argentina and Spain with 270 subjects, who will be followed until 12 months after the end of treatment. The recruitment will begin during the second half of 2016, and the study should be finished in 2018. The primary parameters of evaluation of the effectiveness will be the parasitological response determined by serial PCR obtained at 12 months of treatment (Barreira et al., 2016).

2. POTENTIAL CANDIDATES FOR ANTI-T. CRUZI DRUGS We can follow the process of drug development for Chagas disease in different phases (Salomão et al., 2016): (i) the first phase (1909-1970), during which an extensive list of compounds was subjected to pre-clinical and clinical trials, but their mechanisms of


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trypanocidal action were rarely studied; penicillin, amphotericin B, puromycin, primaquine, the bisquinaldine Bay-7602, metronidazole, piperamide and nitrofurans were used for the treatment chagasic patients, without evident success (Brener, 1979, De Castro, 1993); (ii) the second phase (1970-2005), which began with the clinical use of Nif and Bz, clinical trials with allopurinol, ketoconazole, itraconazole and fluconazole and studies focusing on the mechanism of action of a variety of compounds, such as purine derivatives, nitroheterocycles and azoles (Brener, 1975; Coura, 1996; Urbina, 1999; Coura & De Castro, 2002); and (iii) the third phase (2005), which began with the publication of the T. cruzi genome (El-Sayed et al., 2005), allowing for the generation of transgenic parasites expressing β-galactosidase (Tulahuen lacZ strain) (Buckner et al., 1996), tandem tomato fluorescence protein or the firefly luciferase protein (Y luc and Brazil luc strains) for whole cell-based assays (Hyland et al., 2008; Canavaci et al., 2010; Andriani et al., 2011). A fourth phase is now delineated with the increasing number of drug development programmes involving a network of researchers, non-governmental organizations and pharmaceutical companies (Chatelain & Ioset, 2011; Jakobsen et al., 2011). As stated in Ferreira and coworkers (2016), “... these partnerships have closed important gaps in the drug development chain, leading to significant improvements in the associated technologies and ultimately in the overall drug discovery process.” The advances in screening technologies have resulted in a switch from target-based screening towards phenotypic screens (Gilbert et al., 2011; Salomão et al., 2016), and the development of high-throughput screening (HTS) and high-content screening (HCS) (Bettiol et al., 2009; Engel et al., 2010; Nohara et al., 2010; Carmody et al. 2010a,b; Keenan et al., 2013; Peña et al., 2015; Alonso-Padilla et al., 2015) are allowing for the rapid evaluation of large compound libraries (Ferreira et al., 2016). In addition, the development of bioluminescent T. cruzi also allows for the analysis of infection in experimentally infected mice (Hyland et al., 2008; Andriani et al., 2011; Calvet et al., 2014; Lewis et al., 2015; Francisco et al., 2015). If combined, the major HTS campaigns for Chagas disease have tested more than 2.5 million compounds (Moraes & Franco, 2016).

2.1. Ergosterol Synthesis Inhibitors Because T. cruzi has a strict requirement for specific endogenous ergosterol, enzymes of sterol metabolism have been studied as potential drug targets for more than twenty years (Urbina, 2009; Buckner & Urbina, 2012; Macedo-Silva et al., 2015; Bermudez et al., 2016). The most studied is C14α-sterol demethylase (CYP51), a cytochrome P450 enzyme that catalyses the removal of the C14 methyl group, leading to the accumulation of 14amethylsterols (Lepsheva et al., 2011; Yu et al., 2015).

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Figure 2. Azolic TcCYP51 inhibitors: (a) posaconazole; (b) E1224; (c) VNI (Lepesheva et al., 2010); (d) VFV (Lepesheva et al., 2015); (e) VT-1161 (Hoekstra et al., 2015); (f) NEU321 (Andriani et al., 2013); (g) a bromophenoxy imidazole (Gunatilleke et al., 2012, cpd 1); (h-j) halogenated biphenyl imidazoles (Suryadevara et al., 2013, cpds 36j, 36k, & 36p); (k) a piperazinyl-carbamate imidazolyl-2phenylethanol (De Vita et al., 2016, cpd 7).

Studies of CYP51 inhibitors began in the 1980s with azoles, a class of five-membered heterocyclic compounds of nitrogen; those with 2, 3 and 4 N atoms are called imidazoles, triazoles and tetrazoles, respectively. As trypanocidal agents, the imidazoles miconazole and econazole (Docampo et al., 1981), ketoconazole, the 1,2,4-triazoles fluconazole and


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itraconazole (McCabe et al., 1986; Urbina et al., 1988; Buckner et al., 2003; Moreira-da-Silva et al., 2012), the bistriazole ICI-195,739 and its D(+) isomer - D-0870 (Lazardi et al., 1991; Maldonado et al., 1993; Urbina et al., 1996), and the 1,2,4-triazoles voriconazole (Gulin et al., 2013), albaconazole (Urbina et al., 2000; Diniz et al., 2010), TAK-187 (Urbina et al., 2003a; Corrales et al., 2005), posaconazole (Urbina et al., 1998; Molina et al., 2000; Urbina, 2009) and ravuconazole (Urbina et al., 2003b; Diniz et al., 2010) have been studied. Two phase II clinical trials with chronic patients treated with posaconazole (StopChagas and Chagasazol) (Figure 2a) (Molina et al., 2014) and one trial of E1224 (Figure 2b) (Torrico, 2013) failed to maintain a sustained response during follow-up, indicating insufficient efficacy as monochemotherapy. Posaconazole was also used in the treatment of a chronic chagasic patient with systemic lupus erythaematosus, completely eliminating T. cruzi (negative PCR 12 months after treatment), whereas Bz was unable to clear the circulating parasites (Pinazo et al., 2010). More recently, new series of azoles and no-azolic compounds inhibitors of CYP51 have been assayed against T. cruzi. Tipifarnib, an inhibitor of the human protein farnesyltransferase and an anti-cancer drug candidate, was able to inhibit amastigote proliferation and the activity of TcCYP51 (Hucke et al., 2005). In sequence, analogues were synthesized, and some imidazoles were active in vitro and in vivo, but changes in the tipifarnib scaffold are needed to optimize the pharmacokinetic properties of this series (Kraus et al., 2009, 2010; Buckner et al., 2012). The carboxamide-containing β-phenyl-imidazole VNI (Figure 2c), identified from a collection of azoles from Novartis (Lepesheva et al., 2010), was active in acute and chronic mouse models (Villalta et al., 2013); however, in immunosuppressed animals, no complete parasitological clearance was achieved (Soeiro et al., 2013). In subsequent work, VFV (Figure 2d), a fluoro analogue of VNI, was identified, which demonstrated high in vitro activity and cured the experimental infection with 100% efficacy, displaying oral bioavailability, low offtarget activity, and favourable pharmacokinetics and tissue distribution (Lepesheva et al., 2015). VT-1161, a 1-tetrazole-based drug (Figure 2e) undergoing phase II antifungal clinical trials, is a potent inhibitor of TcCYP51 with in vitro and in vivo activities against T. cruzi. VT1161 was structurally characterized in a complex with TcCYP51, allowing for the optimization of new tetrazole-based analogues, and it possesses good pharmacokinetic properties and an excellent safety profile (Hoekstra et al., 2015). Based on structural motifs identified through two HTS assays (Andriani et al., 2011; Gunatilleke et al., 2012), new imidazoles were prepared, and the diaryl ether NEU321 (Figure 2f) was selected due to its high activity against T. cruzi, its selectivity index (SI) of 159 and its high ligand efficacy to TcCYP51 (Andriani et al., 2013). The target-based HTS also identified a bromophenoxy imidazole (Figure 2g) with binding affinity to the enzyme and trypanocidal activity at the nanomolar level that supresses sterol biosynthesis in amastigotes more efficiently than posaconazole (Gunatilleke et al., 2012). Among a series of dialkyl imidazoles screened as TcCYP51 inhibitors, a derivative substituted with a biphenyl-3-amine group in the imidazole moiety was selected, and in subsequent in vivo assays, it led to high levels of parasitological cure (Suryadevara et al., 2009). Due to its high hydrophobicity, this hit was optimized, leading to three halogenated derivatives (Figure 2h-2j) that were active in vitro against T. cruzi and displayed suitable physicochemical properties (Suryadevara et al., 2013). Friggeri and coworkers (2013, 2014) synthesized imidazolyl-2-phenylethanol derivatives, and several of them were active against intracellular

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amastigotes and inhibited TcCYP51. In sequence, eight new derivatives were prepared and assayed against the parasite, and the most active was a piperazinyl-carbamate derivative (Figure 2k) with nanomolar IC50 values, low cytotoxicity and good chemical and metabolic stability (De Vita et al., 2016). From drug screening targeting Mycobacterium tuberculosis CYP51 (Podust et al., 2007), 4-aminopyridyl-based derivatives were identified as non-azolic inhibitors of T. cruzi´s enzyme. LP10, the lead compound (Figure 3a), binds tightly to TcCYP51, is active against intracellular amastigotes (Chen et al., 2009), displays curative effects in vivo and blocks the 14-αdemethylation step in the parasite (Doyle et al., 2010). LP10, which is a mixture of S- and Risomers, has been optimized, yielding a halogenated bi-aryl R isomer (Figure 3b) that inhibited T. cruzi proliferation at picomolar concentrations (Choi et al., 2014). These compounds were also evaluated in a mouse model of infection with T. cruzi (Y luc) (Calvet et al., 2014). Additionally, from the N-arylpiperazine series of this class of inhibitors, two chlorinated compounds with high selectivity and potency emerged (Figures 3c,3d); in vivo, these compounds led to almost complete suppression of parasitaemia (Vieira et al., 2014).

Figure 3. Non-azolic TcCYP51 inhibitors: (a) LP10 (Chen et al., 2009); (b) an R isomer bi-aryl analogue of LP10 (Choi et al., 2014, cpd (R)-4); (c, d) chlorinated N-arylpiperazine derivatives (Vieira et al., 2014a, cpds 9 & 10).

2.2. Cruzain Inhibitors Cruzain (cruzipain or gp51/57) is a cathepsin-L-like protease of the papain family in T. cruzi, which is responsible for the major proteolytic activity of all life-cycle stages and is essential for parasite intracellular replication and differentiation (Eakin et al., 1992; McKerrow


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et al., 2009); it is also a validated drug target for Chagas disease chemotherapy (Cazzulo et al., 2001; Duschak, 2011; Doyle et al., 2011; Branquinha et al., 2015). Using quantitative structureactivity relationships, X-ray crystallography, virtual and library screenings and lead optimization, new scaffolds of cruzain inhibitors have been identified (McGrath et al., 1995; Martinez-Mayorga et al., 2015). Based on the nature of the interaction with cruzain’s active site, enzyme inhibitors have been classified as irreversible, forming covalent bonds with cysteine sulfur, and as reversible, forming 1,2-adducts with cysteine that are generally unstable (Nicoll-Griffith, 2012). Different classes of irreversible inhibitors of cruzain have been studied (Steverding et al., 2006), such as allyl sulfones (Götz et al., 2004; Fennell et al., 2013), vinyl sulfonamides (Roush et al., 2001), dipeptidyl epoxyesters (Gonzalez et al., 2007), vinyl sulfones (Jaishankar et al., 2008; Kerr et al., 2009; McKerrow, 1999; Sajid & McKerrow, 2002) and vinyl-sulfonecontaining macrocycles (Chen et al., 2008). The vinyl sulfone cruzain inhibitor K777 (Figure 4a) was active in vitro against Nif- and Bz-resistant strains of T. cruzi and also in vivo in mouse and dog models (Engel et al., 1998; Doyle et al., 2007; Barr et al., 2005; McKerrow et al., 2009). The arginine K777 analogue WRR-483 (Figure 4b) is an effective cysteine protease inhibitor with in vitro and in vivo activity similar to that of K777; crystallographic studies confirmed that its mode of action involved targeting the active site of cruzain (Chen et al., 2010). The oxyguanidine analogues WRR-666 and WR-669 (Figures 4c, d), bind noncovalently to cruzain with potency similar to previous vinyl sulfone inhibitors that have been studied (Jones et al., 2015). The development of K777 by the Institute for One World Health (iOWH) was halted in 2005 due to “hepatotoxicity and manufacturing problems” (Sajid et al., 2011; Sterveding, 2015). Since then, the Sandler Center (University of California), in association with National Institute of Allergy and Infectious Diseases (NIAID), has conducted safety studies, and in 2009, DNDi joined forces to obtain funds to complete the Investigational New Drug (IND) filing for K777 (McKerrow et al., 2009; Sajid et al., 2011). In 2013, the DNDi Scientific Advisory Committee recommended interruption of this project due to tolerability findings at low doses in primates and dogs (DNDi, 2014). Among reversible inhibitors, we found bis-arylacylhydrazides (Li et al., 1996), aryl ureas (Du et al., 2002) and ketone and α-ketoester-based compounds (Huang et al., 2002; Choe et al., 2005). Another group of compounds that has been studied as cruzain reversible inhibitors are those containing a nitrile head: purine nitriles (Figure 5a) (Mott et al., 2010); nitrile analogues of odanacatib, a cathepsin K inhibitor (Figures 5b, c) (Beaulieu et al., 2010; Ndao et al., 2014); and dipeptidyl nitriles (Figure 5d) (Avelar et al., 2015). Among non-peptidyl inhibitors of cruzipain, compounds of different classes were active against T. cruzi: (i) thiosemicarbazones (Figures 5e-i) (Du et al., 2002; Greenbaum et al., 2004; Siles et al., 2006; Leite et al., 2006; Caputto et al., 2011; Fonseca et al., 2015; Espíndola et al., 2015; Costa et al., 2016); (ii) thiazolylhydrazones and thiazolidinones (Figures 5j-l) (Leite et al, 2007; Hernandez et al., 2010; Oliveira-Filho et al., 2015; Gomes et al., 2016); (iii) oxadiazoles (Figure 5m) (Santos-Filho et al., 2009, 2012); (iii) aryl-oxymethyl ketones (Figure 5n) (Brak et al., 2008, 2010; Neitz et a., 2015a); (iv) acetamide benzmidazoles (Figure 5o) (Ferreira et al., 2014); and (v) oxorhenium (V) and palladium (II) complexes (Figure 5p) (Fricker et al., 2008). Ligand- and target-based methods were employed to screen non-peptidic non-covalent cruzain inhibitors using the ZINC database. From 23 compounds assayed as cruzain inhibitors, 12 hits with affinity for the enzyme in the micromolar range were selected. The most potent

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compounds were assayed against trypomastigotes of the Tulahuen lacZ strain, revealing Nequimed42, which was 10 times more active than Bz, and identifying the 2acetamidothiophene-3-carboxamide group as essential for enzyme and parasite inhibition activities. Employing the strategy of molecular simplification, a smaller compound was obtained: Nequimed176 (Figure 5q). Non-covalent binding to cruzain was confirmed by determining the binding mode of this compound through X-ray crystallographic studies (Wigglers et al., 2013).

Figure 4. Irreversible cruzain inhibitors: (a) K777 (Engel et al., 1998); (b) WRR-483 (Chen et al., 2010); (c) WRR-666; and (d) WR-669 (Jones et al., 2015).

During the search of cruzain inhibitors, several compounds, although active against the parasite, showed no effect on cruzain: 1-indanone thiazolylhydrazones such as TZH9, inhibiting ergosterol biosynthesis (Figure 6a) (Caputto et al., 2012) and thiazolidinones (Figure 6b) (Moreira et al., 2014). Additionally, a 4-pyridyl analogue of K777 was identified that was 10 times more active than the lead compound, inhibiting primarily CYP51 (Figure 6c) (Choy et al., 2013).


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Figure 5. Reversible cruzain inhibitors: (a) purine nitrile (Mott et al., 2010, cpd 32); (b, c) nitrile analogues of odanacatib (Beaulieu et al., 2010, cpds 26; Ndao et al., 2014, Cz007); (d) dipeptidyl nitrile (Avelar et al., 2015, cpd 5); (e, f) 1-indanone thiosemicarbazones (Caputto et al., 2011, cpd 12); (g, h) aryloxy thiosemicarbazones (Espíndola et al., 2015, cpds 30 & 31); (i) thiosemicarbazone (Costa et al., 2016, LpQM-19); (j) 4-oxothiazolylhydrazone (Leite et al, 2007, cpd 6f); (k) 4-thiazolidinone (Oliveira-Filho et al., 2015, cpd 20); (l) 1,3-thiazole (Gomes et al., 2016a, cpd 1c); (m) oxadiazole (Santos-Filho et al., 2012, cpd 6d); (n) aryl-oxymethyl ketone (Brak et al., 2008, cpd 54); (o) acetamide benzimidazole (Ferreira et al., 2014, cpd 8d); (p) oxorhenium (V) complex (Fricker et al., 2008, cpd 14); (q) Nequimed176 (Wigglers et al., 2013).

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Figure 6. Compounds active against T. cruzi but not acting as cruzain inhibitors: (a) a 1-indanone thiazolylhydrazone (Caputto et al. 2012, TZH9); (b) a thiazolidinone (Moreira et al., 2014, cpd 4h); (c) a 4-pyridyl analogue of K777 (Choy et al., 2013 cpd 4).

2.3. Nitroheterocyclics Nitrocompounds are usually avoided in medicinal chemistry programmes because the presence of a nitro group creates concerns regarding toxicity issues associated with DNA damage, but at the same time, this functional group is usually involved in the desired biological activity (Walsh & Miwa, 2011; Patterson & Wyllie, 2014; Keenan & Chaplin, 2015). The introduction of Nif/eflornithine combination therapy for the treatment of human African trypanosomiasis (HAT) and the rediscovery of fexinidazole (Torreele et al., 2010), which is undergoing clinical trials for HAT (Steinmann et al., 2015) and visceral leishmaniasis (Sundar & Chakravarty, 2015) rekindled interest in nitrocompounds as chemotherapeutic candidates. Regarding Chagas disease, fexinidazole was evaluated in mouse models of acute and chronic infection, leading to high cure rates and reduced myocarditis (Bahia et al., 2012, 2014a; Caldas et al., 2014). Subsequently, a phase II clinical trial was performed in chronic chagasic patients in Bolivia using fexinidazole treatment (NCT02498782), and it was observed that parasitaemia was cleared; however, after recruiting 47 participants, some safety and tolerability issues arose, and it was decided to conclude the trial without the inclusion of new participants (DNDi, 2016). In collaboration with DNDi, 3-nitro-1H-1,2,4-triazoles-based aromatic and aliphatic amines, piperazines, amides and sulfonamides were assayed by Papadopoulou and co-workers. Several triazoles displayed activity against intracellular amastigotes at nanomolar levels with SI > 200, whereas the removal of the nitro group led to inactive compounds (Figures 7a-g) (Papadopoulou et al., 2011, 2012, 2013a). Selected nitrotriazoles with good ADMET and not mutagenic in the Ames test were assayed in infected mice (T. cruzi Y luc strain) (Andriani et al., 2011). By live imaging techniques, the most effective compounds in reducing the parasite index (PI) (luciferase signal ratio after/before treatment) were a 3-nitrotriazole-based amide (Figure 7h) and a thiophene sulfonamide (Figure 7i) with no detectable parasite signal (Papadopoulou et al., 2013b). In the same line of investigation, 3-nitrotriazole-based piperazides were synthesized, and most of them were potent and selective against T. cruzi, with good ADMET characteristics, also being substrates of TbNTR and TcNTR. In vivo, the dichlorophenyl piperazide derivative (Figure 7j) presented a performance similar to that of Bz (Papadopoulou et al., 2015b). The synthesis by the same research group of 5-nitro-2aminothiazoles-based amides led to a piperazine amide (Figure 7k) that was 3.9-fold more active in vitro than Bz, but it did not activate TbNTR (Papadopoulou et al., 2016).


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Based on the structure of Nif, a range of hydrazones of 5-nitro-2-furaldehyde with adamantane alkanohydrazides were synthesized, and the most active was a carbohydrazone with a nanomolar IC50 against trypomastigote forms and an SI of 55. The trypanocidal activity of this series was associated with increased lipophilicity and the presence of a nitro group (Figure 7l) (Foscolos et al., 2016).

Figure 7. Nitroheterocyclics active against T. cruzi. 3-nitrotriazole derivatives active in vitro against T. cruzi: (a) aromatic amine (Papadopoulou et al., 2011, cpd 17); (b) aliphatic amine (Papadopoulou et al., 2011, cpd 29); (c) amide (Papadopoulou et al., 2012, cpd 13); (d) sulfonamide (Papadopoulou et al., 2012, cpd 35); and (e-g) piperazines (Papadopoulou et al., 2011, cpd 42, 2013a, cpds 3 & 7). 3nitrotriazole derivatives active in vivo against T. cruzi: (h) amide (Papadopoulou et al., 2013b, cpd 4), (i) thiophene sulfonamide (Papadopoulou et al., 2013b, cpd 11); and (j) dichlorophenyl piperazide (Papadopoulou et al., 2015b, cpd 3). (k) 5-nitro-2-aminothiazole-based piperazine amide (Papadopoulou et al., 2016, cpd 6); (l) nitro adamantane acetic hydrazide (Foscolos et al., 2016, cpd 4k).

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2.4. Repositioning Campaigns, Combinations of Drugs and Dual Targeted Compounds Currently, two approaches have commonly been used in the development of drugs for neglected diseases: repositioning and combination. New uses for known drugs constitute an attractive approach because their safety and pharmacokinetic profiles have already been optimized for human use, and manufacturing and stability issues have already been addressed (Aubé, 2012; Kaiser et al., 2015). Drug repositioning is based on both the “promiscuous” nature of a drug, which could interact with multiple targets, and the ability of the targets relevant to a particular disease to play critical roles in other biological processes (Ekins & Williams, 2011; Sardana et al., 2011). In recent years, drug repositioning has accounted for approximately 30% of FDA-approved drugs and vaccines (Jin & Wong, 2014). Bromocriptine (antiparkinson and antidiabetic drug) (Figure 8a), amiodarone (antiarrhythmic drug) (Figure 8b) and levothyroxine (hypothyroidism drug) (Figure 8c) were selected in a screening campaign for cruzain inhibitors of the DrugBank database, followed by assays with the enzyme and with T. cruzi epimastigotes (Bellera et al., 2013, 2014). In a subsequent study, virtual screening of the Merck Index 12th database, followed by assays in vitro and in a mouse model of acute infection, clofazimine (anti-leprosy drug) (Figure 8d) and benidipine (hypertension drug) (Figure 8e) were identified (Bellera et al., 2015). These drugs were also assayed in a chronic model of infection using the myotropic K98 strain of T. cruzi and were shown to reduce the parasite burden and inflammatory processes in cardiac and skeletal muscles at levels superior to those of Bz (Sbaraglini et al., 2016). Based on the results of 268 polyamine analogues previously assayed against T. cruzi, computational models with the predictive ability of discrimination between active and inactive compounds were combined and then applied in a virtual screening in the DrugBank and Sweetlead databases. Thus, 45 approved drugs were selected as anti-T. cruzi candidates, and among them, triclabendazole (antihelmintic drug) (Figure 8f), sertaconazole (skin infections drug) (Figure 8g) and paroxetine (antidepressant drug) (Figure 8h) were shown to inhibit epimastigote proliferation and putrescine uptake by the parasite (Alberca et al., 2016). A collaboration between the University of California San Francisco (UCSF) and the Genomics Institute of the Novartis Research Foundation (GNF) has screened more than 160,000 compounds from the ACL (Academic Collaboration Library, ACL, USA) using an image-based HCS assay (Engel et al., 2010), identifying a series of chemotypes. From the xanthine scaffold, the trifluoromethyl analogue GNF7198 was optimized (Figure 8i), which was effective in a mouse footpad model of acute T. cruzi infection (Canavacci et al., 2010). However, due to the complexity and high costs of the synthesis of xanthine analogues, the series was abandoned (Neitz et al., 2015b). Screening of 700 FDA-approved drugs has revealed a variety of compounds with in vitro activity against T. cruzi, such as clemastine (antihistaminic) (Figure 8j) and amlodipine (calcium channel blocker) (Figure 8k). Although clemastine (5 or 100 mg/kg/day - mpk) or amlodipine (10 mpk) administration alone to T. cruzi-infected mice (Tulahuen strain) did not interfere with the course of infection, the clemastine/posaconazole combination led to the survival of 75% of the animals; amlodipine/posaconazole almost totally suppressed parasitaemia, and the survival rate was 80-100% (Planner et al., 2014).


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Figure 8. Drugs active against T. cruzi obtained from repositioning strategies: (a) bromocriptine; (b) amiodarone; (c) levothyroxine; (d) clofazimine; (e) benidipine; (f) triclabendazole; (g) sertaconazole; (h) paroxetine; (i) GNF7198; (j) clemastine; (k) amlodipine; (l) pyronaridine; (m) azelastine; (n) ifenprodil; (o) ziprasidone; (p) clofibrate; (q) mebeverine; (r) tadalafil.

Data from the literature and from HTS screenings for T. cruzi from the Broad Institute (MIT) were collected and used by Ekins and coworkers (2015) to build Bayesian models to predict in vitro activity. These models were used to score several small libraries of molecules, selecting 97 molecules for assays using intracellular amastigotes (Engel et al., 2010), and those

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with IC50 values less than 10 μM were assayed in vivo using T. cruzi Brazil luc strain35. In this study, pyronaridine (antimalarial drug) (Figure 8l) showed an efficacy of 85.2%, emerging as the lead compound for further optimization (Ekins et al., 2015). A cascade screening of 963 compounds (NIH and Selleck-Chem libraries) was developed using, first, a single-point HCS for intracellular amastigotes (DNA staining) (Engel et al., 2010), followed by similar assays of dose- and time-dependent trypanocidal effects using higher parasite loads and a final CYP51 test to remove compounds acting on this enzyme. This study identified azelastine (histamine antagonist) (Figure 8m), ifenprodil (inhibitor of the NMDA receptor) (Figure 8n), ziprasidone (antipsychotic drug) (Figure 8o) and clofibrate (lipid-lowering drug) (Figure 8p) as anti-T. cruzi candidate drugs. Their activity, together with good pharmacokinetic parameters, makes them attractive starting points for lead optimization (De Rycker et al., 2016). Kaiser and coworkers (2015) tested 100 registered drugs selected for their repositioning potential against trypanosomatids and Plasmodium falciparum. For T. cruzi, in vitro screening was performed against intracellular amastigotes (Tulahuen lacZ strain) (Orhan et al., 2010), and in addition to nitrofurane derivatives and azoles, two other drugs were highlighted: the phenylbenzoate mebeverine (antispasmodic drug) (SI=18) (Figure 8q) and tadalafil (SI=26) (erectile dysfunction drug) (Figure 8r). The authors considered that the trypanocidal activity was moderate, but their potential use for repositioning in Chagas’ disease must be further evaluated (Kaiser et al., 2015). The main advantages of drug combinations are the possibility of reducing doses and treatment duration, as well as reducing collateral effects and resistance development. In vivo studies using acute murine models of T. cruzi infection have demonstrated that the combination of drugs, even if not leading to a parasitological cure, could reduce parasite burden, mortality rates and tissue lesions. In 1993, different combinations of sterol inhibitors were assayed, and it was observed that treatment for 7 days with ketoconazole (Figure 9a) (15 mpk) plus terbinafine (100 mpk) (Figure 9b) led to a 66% parasitological cure (subinoculation of organs in naïve animals) and to 100% survival, whereas each drug alone at the same doses led to survival percentages of 66% and 0%, respectively (Maldonado et al., 1993). Additionally, ketoconazole (15 mpk)/lovastatin (20 mpk), at doses at which the former alone offered only limited protection, yielded 100% survival of the animals (Urbina et al., 1993). Later, several studies associating Bz and other drugs were performed in acute models of infection, using parasite strains with different susceptibilities to this standard drug. It was observed that the combination of Bz/ketoconazole was beneficial in infections due to strains susceptible (CL) or moderately resistant (Y) to Bz. However, Bz (25 mpk) or ketoconazole (120 mpk) for 20 days cured none of the animals; when combined, the cure rates (haemoculture and xenodiagnoses) were 38.5 and 71.4% for the Y and CL strains, respectively. None of the animals infected with the resistant Colombiana strain with different combinations were cured (Araujo et al., 2000). The glutamate analogue BSO (L-buthionine(S,R)-sulfoximine) (Figure 9c) (200 mpk), when combined to Nif (2.5 mpk) after treatment for 20 days, led to increased survival rates and decreased cardiac parasite burden in mice infected with the clone Dm28c (Faundez et al., 2008).


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Figure 9. Drugs used in combination in in vivo models of T. cruzi-infection: (a) ketoconazole; (b) terbinafine (Maldonado et al., 1993); (c) BSO (Faundez et al., 2008); (d) itraconazole (Martins et al., 2015); (e) DB289 (Batista et al., 2011); (f) DB766 (Batista et al., 2011); (g) DB1965 (Silva et al., 2012); (h) 35DAP073 (Guedes-da-Silva et al., 2016); (i) tetrahydro-β-carboline (Valdez et al., 2012, cpd C4); (j) clomipramine (Strauss et al., 2013; Garcia et al., 2016); (k) curcumin (Novaes et al., 2016).

When used for 10 days, which is considered a suboptimal period, Bz (100 mpk) or posaconazole (20 mpk) cured none of the infected animals. In contrast, in acute infections, the combination Bz100/Pos20 led to cure percentages of 50% (Y strain) and 66.7% (Tulahuen) in acute infections and 16.7% (Tulahuen) in chronic infections, showing that combinations for shorter periods than usually used led to cures of a proportion of the animals (Cencig et al., 2012).

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The combination of Bz with sterol synthesis inhibitors was also assayed in acutely infected mice (Y strain) treated for 20 days by the group of Bahia (Bahia et al., 2014b). Although itraconazole (75 mpk) (Figure 9d) and Bz75 led to cure rates of 0% and 20% (immunosuppression and PCR), respectively, the combined treatment led to a cure percentage of 80%. This combination also reduced the parasite burden in uncured animals (Martins et al., 2015) and resulted in less elimination of Bz (prolonged Bz half-life) (Moreira-da-Silva et al., 2012). In experiments with the VL-10 strain, which is highly resistant to Bz, no cure was observed by treatment with Bz100 or Pos40, whereas the combination Bz100/Pos20 cured 2030% of the animals (Diniz et al., 2013). The group of Soeiro investigated different schemes of combination of Bz with aromatic diamidines (DAs) and aylimidamides (AIAs) (Soeiro et al., 2013). Treatment for 20 days with the diamidine DB289 (25 mpk) (Figure 9e) led to an 85% survival rate, whereas when combined with Bz50, the survival rate was 100%, in addition to an observed reduction in tissue lesions. However, no parasitological cure (haemoculture and PCR) was achieved (Batista et al., 2011). Using the same protocol, the combination of DB766 (Figure 9f) (50 mpk) with Bz50 led to 100% survival, whereas for the untreated, AIA alone and Bz groups, the respective values were 0%, 50% and 80%. No parasitological cure was observed by treatment with a single drug, and Bz/DB766 led to therapeutic failure in 87% of the animals (Batista et al., 2011). Treatment for 20 days with the AIA DB1965 (Figure 9g) (5 mpk) combined with Bz50 led to 100% survival, although no parasitological cure was achieved (Silva et al., 2012). The combination of the AIA 35DAP073 (0.5 mpk) (Figure 9h) with Bz100 for 10 days in mice infected with the Colombiana strain resulted in the suppression of parasitaemia, the elimination of neurological toxic effects, the survival of all of the animals, and a 60-69% reduction in the parasite load (qPCR), compared to that achieved with Bz or the amidine alone (Guedes-da-Silva et al., 2016). The combination of Bz5 with a tetrahydro-β-carboline compound (C4) (5 mpk) (Figure 9i) in acutely infected mice (Y strain) treated for 17 days led to survival of all of the animals and a drastic decrease in cardiac inflammatory infiltrates and parasite burden, similar to the treatment with only Bz100 (Valdez et al., 2012). The combination of Bz50 with clomipramine (5 mpk; Clo5) (Figure 9j) in acutely infected mice (Tulahuen strain) treated for 30 days led to survival of 90-100% of the animals, whereas for Clo5 alone, this percentage decreased to 65%, with results comparable to those of Bz100 (Strauss et al., 2013). In a subsequent study, a 14-day combined treatment of Bz25 and clomipramine (2.5-12.5 mpk) led to the total suppression of parasitaemia; all of the animals survived, and in the case of chronic infection, cardiac damage and inflammation decreased (Garcia et al., 2016). The plant polyphenolic curcumin (100 mpk, C100) (Figure 9k) was combined with Bz50 or Bz100 to treat mice infected with T. cruzi (Y strain) for 20 days. Curcumin potentiated the efficacy of Bz, reducing the cardiac parasite load, inflammation and IFN- seric levels and increasing cure rates. Using this association, it was possible to prevent recrudescence (immunosuppression, haemoculture and PCR) after completion of the treatment, even with the suboptimal dose of Bz50. The percentages of recrudescence were: 75% for C100, 66.7% for B100, 25% for B50 and 0% for Bz50/C100 and Bz100/Cur100 (Novaes et al., 2016). Bustamante and coworkers (2014) demonstrated in different mouse models that shorter or intermittent treatment and drug combinations could increase the rates of parasitological cure, circumventing the toxicity of Bz and Nif. For the CL strain (Bz-susceptible), Bz10/Pos10 (10 days) led to cures for 66% of animals in the acute phase, whereas Bz20 alone (20 days) and


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Bz10/Allo10 (10 days) led to only 10 and 20% cure rates, respectively. For the Brazil strain (Bz-susceptible), a 100% cure rate was accomplished with only the treatment of Pos5 (5 days), followed by Bz27 (7 intermittent doses on a 5-day interval).

Figure 10. Dual target compounds active against T. cruzi: (a, b) heteroallyl-containing 5-nitrofurane and a thia-analogue (Gerpe et al., 2008, cpd 8) Gerpe et al., 2009, cpd 8); (c, d) 3-nitrotriazole-based amides (Papadopoulou et al., 2015a, cpds 2 & 5); (e, f) 3-nitrotriazole-based carbinols (Papadopoulou et al., 2015a, cpds 18 & 19); (g, h) halogenated 3-nitrotriazole-base aryloxy-phenylamides (Papadopoulou et al., 2015c, cpds 3 & 4); (i) a nitro acylhydrazone-oxadiazole (Serafim et al., 2014, cpd 6); (j) bis-(furanyl-allylidene)cyclohexanone (Aguilera et al., 2016, cpd 7); (k, l) chromenylphenoxy-naphthalenedione derivatives (Belluti et al., 2014, cpds 3 & 5).

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Multitarget drug design is an alternative strategy to combine drugs involving molecular hybridization of different bioactive pharmacophoric moieties, thus improving the likelihood of efficacy and preventing drug-resistance development (Morphy & Rankovic, 2009; Njogu & Chibale, 2013). Multitarget drug discovery and polypharmacology offer new paradigms with the potential to overcome the limitations of classic “one target, one drug” strategies (Bolognesi & Cavalli, 2016; Talevi, 2016). Heteroallyl-containing 5-nitrofuranes and thia-analogues were synthesized, aiming to promote oxidative stress (reduction of the nitro group) and to inhibit sterol synthesis because the allylamine terbinafine is a known inhibitor of squalene epoxidase inhibitor, and several compounds were active against intracellular amastigotes at low micromolar concentrations with low toxicity (Figure 10a, b). Oxidative stress was verified by measuring cyanide dependent respiration and the inhibition of squalene epoxidase by highresolution gas-liquid chromatography coupled with mass spectrometry (Gerpe et al., 2008, 2009). 3-Nitrotriazole-based amides, carbinols and aryloxy-phenylamides were designed, with the goal of finding compounds with dual targets that act as substrates for NTR-I (reducible nitrogroup) and as TcCYP51 inhibitors (triazole moiety). From this study two amides, two carbinols, and two halogenated aryloxy-phenylamides emerged (Figures 10c-h) as the most active compounds for intracellular amastigotes (Papadopoulou et al., 2015a,c). All of the nitrotriazoles tested were excellent substrates of TbNTR and TcNTR, metabolizing both enzymes at rates comparable to those of Bz and TcCYP51 inhibitors and exhibiting a high docking score with p-chloro aryloxy phenylamide. When assayed in a mouse model of infection with T. cruzi (Y luc strain) (Andriani et al., 2011), the amides and carbinols led to PI values less than 4% (Papadopoulou et al., 2015a), and aryloxy-phenylamide reduced the parasite burden to undetectable levels (Papadopoulou et al., 2015c). The design of a potential cruzain inhibitor, together (N-acylhydrazone moiety) (Moreira et al., 2009) with a nitric oxide donor (furoxan, 1,2,5-oxadiazole N-oxide (furoxan)), led to the synthesis of a nitro acylhydrazone-oxadiazole N-oxide with both intracellular amastigote proliferation and cruzain activity and good permeability and SI (Figure 10i) (Serafim et al., 2014). Based on previous studies showing that the best inhibitors of the triosephosphate isomerase of T. cruzi (TcTIM) were symmetric molecules (Alvarez et al., 2010), new symmetrical difurylidene ketones were synthesized and assayed against epimastigote forms, and the most active were evaluated in mice infected with the CLBrener clone of T. cruzi. A bis-(furanylallylidene)cyclohexanone (Figure 10j) led to a significant reduction in parasitaemia and 100% survival of the animals. By enzymatic and molecular docking, it was shown that this derivative inhibited TcTIM at nanomolar levels and was also a potent cruzain inhibitor (Aguilera et al., 2015). In the search for glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/trypanothione reductase (TR) inhibitors and based on 2-phenoxy-1,4-naphthoquinone, which was previously shown to inhibit TbGAPDH and to interfere with the respiratory chain (Pieretti et al., 2012) and TR (Lizzi et al., 2012), two series of quinone-coumarin hybrids were synthesized. Against T. cruzi, two chromenyl-naphthalenedione derivatives displayed EC50 values similar to Bz, but the SI was very low (Figures 10k, l) (Belluti et al., 2014).


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CONCLUSION Chagas disease cannot be eradicated due to the existence of infected wild triatomines in permanent contact with domestic cycles; however, it is possible to produce a dramatic reduction of burden of the disease by the prevention of transmission and the timely diagnosis and treatment of affected individuals (Sosa-Estani & Segura, 2015). Control and prevention of T. cruzi infection in endemic countries involve sustaining surveillance and control programmes of vectorial transmission and systematic screening in blood banks and organ donation programmes (Dias, 2015; Pinazo & Gascon, 2015). Another worldwide challenge is the detection of infected pregnant women and congenital cases (Carlier et al., 2011; SorianoArandes et al., 2016). In the Amazon basin, the implementation of good manufacturing practices in food preparation and the cooperation of health personnel and the participation of at-risk communities to ensure continuous epidemiological surveillance are also fundamentally required (Coura & Junqueira, 2012, Barbosa et al., 2015; WHO, 2015). In addition to the control of Chagas disease, other challenges in this field include the standardization of preclinical protocols, the definition of biomarkers to monitor treatment efficacy, the knowledge of the mode of action (molecular targets) and the pharmacokinetic and bioavailability characteristics of Nif and Bz. The scenario of drug development for Chagas disease has been transformed in recent years, with the development of the transgenic parasites HTS and HCS bringing a switch from targetbased screening towards whole-parasite-based assays that allow for issues such as host cell permeability and toxicity to be monitored (Bettiol et al., 2009; Andriani et al., 2011; Sykes & Avery, 2013; Alonso-Padilla et al., 2015; Peña et al., 2015). The number of players in a variety of collaborative networks in this scenario is increasing each day (Chatelain & Ioset, 2011; Jakobsen et al., 2011; Chatelain, 2015). In a review of the literature in 2002, we stated that “…. this ideal drug does not exist and possibly it will take a long period of time to be obtained” (Coura & De Castro, 2002). As cited by Moraes and Franco in 2016, millions of compounds have been assayed in HTS campaigns. Unfortunately, despite the great advances in technology and in the knowledge of T. cruzi and infection development, the picture remains grim, and in the near future, we envisage only two routes: new schemes of the old drugs that reduce the doses and lengths of treatment and different combinations of Bz or Nif with azoles, as is the priority of a new study in development by DNDi (DNDi, 2015; Barreira et al., 2016).

ACKNOWLEDGMENTS We would like to thank Gevânio Oliveira-Filho for helpful discussions. This research was funded by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and by Fiocruz.

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