Solange L de Castro & Maria de Nazaré C. Soeiro
Laboratory of Cell Biology, Oswaldo Cruz Institute, FIOCRUZ
Chagas disease, caused by Trypanosoma cruzi, was discovered in 1909 by Brazilian physician Carlos Chagas (1879-1934) (Andrade et al., 2011; Dias, 2015). This disease estimated global prevalence was 7 million people in 1960, 16-18 million in 1990, 9.8 million in 2006, and 5-7 million in 2010 (Schofield et al., 2006; PAHO, 2010; WHO, 2015). The dramatic decrease in its prevalence observed from 1990 to 2010 was primarily a development consequence of transnational programs in Latin America focused on the domestic vectors elimination and blood donor screening: the Southern Cone Initiative, the Andean Countries Initiative, the Central America and Mexico Initiative, and the Amazonian Countries Initiative, all supported by the Pan American Health Organization/World Health Organization (PAHO/WHO) (Schofield et al, 2006; Guhl, 2007; Dias, 2009; Moncayo & Silveira, 2009).
Chagas disease is transmitted to humans by triatomine vectors, infected blood transfusion, oral and congenital transmission and less commonly by direct transmission from T. cruzi reservoirs, ingestion of undercooked 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). As the vector control and blood banking programs success, congenital (Gebrekristos & Buekens, 2014; Carlier et al., 2015; Álvarez et al., 2017) and oral (Alarcón-de- Noya et al., 2010; Shikanai-Yasuda & Carvalho, 2012; Goés-Costa et al., 2017; Silva-dos-Santos et al., 2017) transmissions have become important sources of Chagas disease new cases. This disease, classically associated with poor and rural populations, underwent the urbanization process in the 1970s and 1980s. The spread to North America, Europe, Asia, and Australia was continuously increasing, creating new epidemiological, economic, social, and political challenges (Schmunis, 2007; Schmunis & Yadon, 2010; Jackson et al., 2014). The Chagas disease prevalence analysis in immigrants from Latin America revealed an infection rate of 4.2% in Europe by 2004, with a large heterogeneity depending on the origin country (Requena-Méndez et al., 2015, 2017).In non-endemic countries, Chagas disease transmission is associated with the congenital route, blood transfusion and organ transplantation (Bern et al., 2011; Rodriguez-Guardado et al., 2015; Antinori et al., 2017). In the United States of America (USA) applying the seroprevalence reported by PAHO (2006) and assuming a transmission rate by infected pregnant women 1-5%, Bern & Montgomery (2009) estimated 300,000 infected persons and 63-316 congenital cases of T. cruzi infection/year. Recently updating this estimate revealed a decrease of about 20% in the infections number, with undocumented immigrants not included, which may lead to about 109,000 additional cases (Manne-Goehler et al., 2016). The Centers for Disease Control and Prevention (CDC) has classified Chagas disease as one of the five neglected parasitic diseases in the USA (CDC, 2015). Europe is home to approximately 3.5 million Latin Americans, with an estimated 90,000 infected individuals, with approximately 50,000 of these in Spain (WHO, 2010; Gascon et al., 2010; Antinori et al., 2017; MongEmaillo et al., 2017).
2. Nifurtimox and benznidazoleThe etiologic treatment for Chagas disease is restricted to two nitroheterocyclic drugs introduced in the 1960-70s: benznidazole (Bz, LAFEPE and Abarax/ELEA) and nifurtimox (Nif, LAMPIT/Bayer) (Fig. 1). Bz was produced until 2003 by Roche (Rochagan® and Radanil®), when its manufacturing was transferred to a pharmaceutical company linked to the Brazilian government, the Pernambuco State Pharmaceutical Laboratory (Lafepe, acronym in portuguese). The drug was prepared with the active ingredient donated by Roche until 2011, when its production was interrupted due to the non-approval of Good Manufacturing Practices (Manne et al., 2012), being resumed after certification by the National Health Surveillance Agency (ANVISA, Brazil). In Argentina, production was initiated by the private laboratory Elea (Abarax®), through an association with the Ministry of Health and the non-governmental organization Mundo Sano Foundation. Currently, Doctors without Borders (MSF) purchases a tablet manufactured by Lafepe (acronym in portuguese) of $0.21 USD while PAHO pays $0.48 for Elea’s product (Pinheiro et al., 2017). Bz was recently approved by the FDA in the US (US Food and Drug Administration) for use in children aged 2-12 years, this being the first treatment for Chagas disease approved in the US (FDA, 2017). Nif Production (Bayer HealthCare) was halted in the 1980s due to demand issues in Brazil, but with the results of positive clinical trials for the human African trypanosomiasis treatment (Alirol et al., 2013), its production resumed in 2000 at Bayer’s plant in El Salvador (Ilopango Bayer). The Nif combination with eflornithine (NECT) as first-line treatment for CNS stage of T. brucei gambiense infection was approved for clinical use and included on the WHO Essential Medicines List. Bayer, its only product, has secured its supply until 2019 and is also developing a new formulation aimed at more individualized treatment (Bayer HealthCare, 2017).
The results obtained with Nif and Bz vary according to the stage of the disease, the period and treatment dose, the age and the geographical origin of the (Coura & De Castro, 2002). Both drugs showed excellent results with high parasitological cure rates during the acute phase, but their efficacy decreases as the infection progresses, thus being crucial for their success, detection and intervention as early as possible (Coura & Borges-Pereira, 2011). The high incidence of side effects, especially in adults, has led to treatment abandonment in many situations (Pinazo et al., 2010; Jackson et al., 2010; Perez-Molina et al., 2012), while children have a higher tolerance to medication (WHO, 2002; Dias et al., 2016). In a recent study with Nif in the US revealed milder side effects, being mostly controlled by medication reduction in dosage and/or temporary suspension (Forsyth et al., 2016).
The Bz and Nif mechanism of action is still being debated, despite these drugs being in clinical use for over 5 decades (DoCampo & Moreno, 1984; DoCampo, 1990; Maya et al., 2003; Sueth-Santiago et al., 2017). Both are considered promedications that require activation by nitroreductases: NTR-I, oxygen-insensitive enzyme that catalyzes the two-electron type reduction of the nitro group, and NTR-II, oxygen-sensitive that catalyzes the one-electron type reduction (Peterson et al., 1979). Nif reduction by NTR-II leads to oxidative stress due to transformation to a nitroanion radical, redox cycling, reactive oxygen species (ROS) generation and lipid peroxidation (DoCampo & Stoppani, 1979; Moreno et al., 1984). Despite in the drugs nitroheterocyclic nature, Bz is a 2-nitroimidazole with lower reduction potential when compared to the 5-nitrofuranic Nif group, thus leading to a lower rate of nitroanion radical formation and reactive species, thus allowing for a better detoxification response by T. cruzi (DoCampo, 1990). Reduction by NTR-I leads to a hydroxylamine intermediate, which undergoes different transformations: in the Nif case, the nitrofuran group is transformed into a highly reactive open-chain unsaturated nitrile (Hall et al, 2011), while with Bz, two consecutive one-electron reductions lead to a dihydroxy-dihydroimidazole derivative, which can break down to glyoxal, a toxic metabolite, which in turn can react with the parasite’s DNA (Hall & Wilkinson, 2012; Patterson & Wyllie, 2014).
3.New drugs development
We can follow the development process of alternative drugs for Chagas disease in different phases (Solomon et al, 2016): (i) first phase (1909-1970), during which an extensive compounds list was subjected to preclinical and clinical trials, with the action mechanism rarely investigated; penicillin, amphotericin B, puromycin, primaquine, the bisquinaldine Bay-7602, metronidazole, piperamide and nitrofurans were used for patient treatment, without evident success (Brener, 1979; De Castro, 1993); (ii) second phase (1970-2005), which starts with the clinical use of Nif and Bz with allopurinol, ketoconazole, itraconazole and fluconazole and studies focused on the action mechanism of compounds wide variety, such as purine derivatives, nitroheterocyclic and azoles (Brener, 1975; Coura, 1996; Urbina, 1999; Coura & De Castro, 2002); (iii) third phase(2005), which starts with the genome of T. cruzi publication (El-Sayed et al, 2005), allowing the transgenic parasites generation expressing β-galactosidase (Tulahuen lacZ strain) (Buckner et al., 1996), tandem tomato fluorescent protein or firefly luciferase (Y luc and Brazil luc strains) for assays with intact parasites (Hyland et al, 2008; Canavaci et al., 2010; Andriani et al., 2011); and (iv) fourth phase with the increasing number of drug development programs involving a researchers network, non-governmental organizations and pharmaceutical companies (Chatelain & Ioset, 2011; Jakobsen et al., 2011). Advances in screening technologies have resulted in a shift from target-based to phenotypic traces (Gilbert et al., 2011; Solomon et al., 2016), and the development of HTS, high-throughput technology and HCS, high-content technology (Bettiol et al., 2009; Engel et al., 2010; Keenan et al., 2013; Peña et al., 2015; Alonso-Padilla et al., 2015) are enabling a large number of compound libraries rapid evaluation (Ferreira et al., 2016). In addition, the bioluminescent T. cruzi development allows for individual analysis of infection in experimentally infected live mice (Hyland et al., 2008; Andriani et al., 2011; Calvet et al., 2014; Lewis et al., 2015; Francisco et al., 2015). Moraes & Franco (2016) assessed that major HTS campaigns have tested over 2.5 million compounds.
Currently, two approaches have been widely employed in drug development for neglected diseases: repositioning and combination. New uses for known drugs are an excellent approach once safety and pharmacokinetic profiles have already been optimized for human use and manufacturing and storage issues have been evaluated (Aubé, 2012; Kaiser et al., 2015). In recent years, repositioning accounts for 30% of products cmo drugs and vaccines approved by the FDA (Jin & Wong, 2014). Combining drugs allows the reduction of doses and treatment time, as well as reducing side effects and the resistance development in parasites. In vivo studies using acute murine infection models have demonstrated that the drugs or candidates combination, even if it does not lead to a parasitological cure, can reduce parasite load, mortality rate, and tissue damage (Maldonado et al., 1993; Urbina et al., 1993a).
An improved understanding of T. cruzi biochemistry and various preclinical approaches have allowed the parasite targets identification such as sterol metabolism, DNA, cysteine protease, nucleotide synthesis and purine salvage pathway, dihydrofolate reductase, glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, and pyrophosphate metabolism. For reviews on targets in T. cruzi, please see Bahia et al. (2014a), Duschak (2016), Rodriguez et al. (2016), Sanchez-Sanchez et al. (2016), Salomão et al. (2016), Menna-Barreto & De Castro (2017), Salomão & De Castro (2017), Sueth-Santiago et al. (2017), Field et al. (2017) and Sales-Junior et al. (2017). Based on the recent literature, we found among the most promising compounds in preclinical studies, azolic and non-azolic compounds acting on C14α-cholesterol demethylase (CYP51), phenoxyphenoxyethyl cyanate derivatives acting on squalene synthase (SQ), different compounds especially thiazoles acting on cysteine protease, DNA ligands and nitrocompounds, more specifically, nitrotriazoles.
3.1. C14α-cholesterol demethylase inhibitors (CYP51): This cytochrome P450 enzyme calyzes the methyl group removal at C14, leading to the 14α-methyl sterols accumulation. As in fungi, since T. cruzi requires endogenous ergosterol, sterol metabolism enzymes have been studied for over 20 years as potential targets for new drugs (Urbina, 2009; Buckner & Urbina, 2012; Macedo-Silva et al., 2015; Bermudez et al., 2016). Most studies on CYP51 inhibitors involve the azoles repositioning, originally developed as antifungal drugs (Lepsheva et al., 2007, 2008; Yu et al., 2015). Azoles are a class of heterocyclic nitrogen compounds; those with 2, 3, and 4 N atoms are called imidazoles, triazoles, and tetrazoles, respectively. Different azoles have shown in vitro and in vivo activity on T. cruzi (Urbina, 2009; Lepsheva et al., 2011; Urbina & McKerrow, 2015).Posaconazol (SCH56592, Noxafil, Schering-Plough) and ravuconazole (RAV, Eisai Co.) (Figs. 2a,b) have an antifungal activity broad spectrum and are generally well tolerated by humans (Morris et al., 2009; Peyton et al., 2015). Posaconazole showed a potent ergosterol synthesis inhibition in T. cruzi, inducing parasitological cure high percentages in acute and infection chronic models, even in the case of Bz-resistant strains of the parasite and in immunosuppressed animals (Urbina et al., 1998; Molina et al., 2000; Urbina, 2009). Posaconazole was also used in the chronic chagasic patient treatment with systemic lupus erythematosus, with negative PCR 12 months post-treatment, while Bz induced a reduction but not elimination of circulating parasites (Pinazo et al., 2010). Ravuconazole led to parasitological cure high percentages in animals in acute phase, except for those infected with Bz-resistant strain (e.g. Colombian) and in chronic models (Urbina et al., 2003a), while in acute canine model, it showed only suppressive effect (Diniz et al., 2010).
Both posaconazole and E1224, a ravuconazole pro-medication (DNDi, 2013) have undergone phase II clinical trials in chronic patients, using PCR negativation as a treatment success endpoint. Briefly: (a) Chagasazole (NCT01162967, Spain) after 40 weeks of posaconazole treatment led to 10-20% negative PCR, while the corresponding value for Bz was 94% (Molina et al, 2014); (b) Stopchagas (NCT01377480, Latin America) evaluated posaconazole, Bz, their combination with negative PCR pergentages of 10, 15.6, 86.7, and 82.1% for the placebo, posaconazole, Bz, and posaconazole+Bz groups, respectively (Stopchagas, 2016; Morillo et al, 2017); and (c) E1224 assay (NCT01489228, Bolivia) after one year of treatment this compound led to 8-31% negative PCR, while Bz reached 81 % (Torrico et al., 2013; DNDi, 2013; Barreira et al., 2016). According to Urbina (2015), treatment failure would be related to E1224 sub-optimal doses and the treatment regimens used.
VNI, a β-phenyl-imidazole containing a carboxamide group (Fig. 2c), identified from a collection of azoles from the Novartis company (Lepesheva et al., 2010), was shown to be active in murine models of acute and chronic infection by the Tulahuen strain (Villalta et al., 2013); however, in animals immunosuppressed with cyclophosphamide after infection with Y or Colombian strains, parasitological cure was not achieved on monotherapy regimens (Soeiro et al., 2013). In subsequent work, VFV (Fig. 2d), a fluoro analog of NIV demonstrated 100 % efficacy in experimental infection, with favorable oral availability and pharmacokinetics (Lepesheva et al., 2015).
Also, VT-1161, a 1-tetrazole-like drug (Fig. 2e) undergoing phase II studies as an antifungal, was shown to be active in vitroand in vivo on T. cruzi. VT-1161 was structurally characterized in a complex with TcCYP51, allowing the new tratrazole analog optimization with good pharmacokinetic properties and an excellent safety profile (Hoekstra et al., 2015).
Furthermore, a recent comparative investigation of two protozoan-specific CYP51 inhibitors, NIV and its derivative VFV, in its derivative VFV, in murine models infected with T. cruzi strain Y showed that different treatment regimens and vehicles, as well as the animal sex can, depending on the compound, have a major impact on cure (Guedes-da-Silva et al., 2017).
The CYP51 inhibitors rational design represents a promising strategy for anti-T.cruzi agents through different approaches (molecular docking, structure-activity correlations, CYP51 inhibition assays, and in vitro and in vivo phenotypic assays). The potency and selectivity of novel pyrazolo[3,4-e][1,4]thiazepine-type CYP51 inhibitors have been investigated being active in vitro on intracellular parasites (Tulahuen strain) and blood forms (Y strain) (Fiuza et al., 2018). In vivo, the compound reduced parasitemia peak by 43 %, but did not lead to survival of the animals. Our results showed that five new analogues were active on intracellular forms, in particular two compounds with selectivity indices (SI) higher than 36, one of these showing activity on trypomastigote forms similar to that of Bz. These results encourage the new pyrazolo[3,4-e][1,4]thiazepine derivatives synthesis in the search for alternative therapies for Chagas disease.3.2. Schalene synthase inhibitors (SQS): This enzyme catalyzes the first step of the two-step reductive dimerization reaction between two farnesyl diphosphate (FPP) molecules to give rise to a squalene molecule (Macedo-Silva et al., 2015). Squalene synthase inhibitors were originally developed for the hypercholesterolemia treatment (Charlton-Menys & Durrington, 2007; Do et al., 2009). The compounds variety activity, including bisphosphonates, benzylamines, and quinuclidines, on mammalian enzymes has also been investigated. 4-Phenoxyphenoxyethylthiocyanate (WC-9) (Fig. 3a) is a potent inhibitor of the T. cruziintracellular amastigote forms proliferation (Cinque et al., 1998; Liñares et al., 2006; Rodriguez et al., 2016a,b) acting as a TcSQS non-competitive competitor in the nanomolar range (Urbina et al., 2003b AAC). WC-9 Analogs were designed and evaluated on T. cruzi intracellular forms, and two fluorinated derivatives (3-(3-fluorophenoxy)- and 3-(4-fluorophenoxy)phenoxyethyl thiocyanate) stood out showing similar activity to the parent compound (Figs. 3b,c) (Chao et al., 2016). Furthermore, Selenium analogues isosteric to WC-9 were synthesized (Fig. 3d) showing activity on intracellular forms at the nanomolar level and with selectivity index (SI) values greater than 900, indicating that the substitution of a thiocyanate group with a selenocyanate led to very potent trypanocidal compounds (Chao et al., 2017).
3.3. Cysteine protease inhibitors
Cruzaine (cruzipain or gp51/57) is a cathepsin-L-like protease of the papine family in T. cruzi, involved in proteolytic activity at all parasite life cycle stages, and is also essential in intracellular differentiation and replication (McKerrow et al., 2009; Cazzulo et al., 2001; Doyle et al., 2011; Ferreira & Andricopulo, 2017). Based on the nature of interaction with the cruzain active site, enzyme inhibitors have been classified as irreversible, forming covalent bond with the cysteine sulfur, and reversible, forming 1,2-type adducts with this amino acid that are generally unstable (Nicoll-Griffith, 2012).Different classes of irreversible cruzain inhibitors have been studied, such as diazomethyl ketones, allyl sulfones, vinyl suffonamides, and vinyl sulfones (McKerrow, 1999; Sajid & McKerrow, 2002; Gonzalez et al., 2007; Jaishankar et al., 2008; Kerr et al., 2009; Chen et al., 2008; Fennell et al., 2013). The vinyl sulfone K777 (Fig. 4a) has been shown to be active in vitroon 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 analog of K777, compound WRR-483 (Fig. 4b) binds weakly to cruzain but is effective in vivo (Chen et al., 2010). The K777 development by the Institute for One World Health (iOWH) was halted in 2005 due to “hepatotoxicity and manufacturing problems” (Sajid et al., 2011; Steverding, 2015; Branquinha et al., 2015). Next, the Sandler Center (University of California), in association with the National Institute of Allergy and Infectious Diseases (NIAID), conducted safety studies, and in 2009, DNDi joined to obtain funding aimed at complementing the K777 analysis (McKerrow et al., 2009; Sajid et al., 2011). In 2013, the DNDi Scientific Committee recommended discontinuation of this project due to tolerability issues at low doses in primate and dogs (DNDi, 2014).
A wide classes variety of compounds showed anti-T. cruzi activity involving cruzain inhibition, such as aryl ureas (Du et al., 2002), purine nitriles (Mott et al., 2010), dipeptidyl nitriles (Avelar et al., 2015), odanacatib analog nitriles (Beaulieu et al., 2010; Burtoloso et al., 2017); thiosemicarbazones (Caputto et al., 2011; Espíndola et al., 2015; Costa et al., 2016), thiazolidinones (Leite et al, 2007; Santos-Filho et al., 2012; Oliveira-Filho et al., 2015), and thiazoles (Cardoso et al., 2014; Gomes et al., 2016; Silva et al., 2017; Oliveira-Filho et al., 2017). For recent reviews on drug-targeted cruzaine, see Martinez-Mayorga et al. (2015) and Ferreira & Andricopulo (2017).
3.4. DNA Binders
Classical aromatic diamidines bind non-covalently and non-intercalably to the DNA minor cleft, and several hypotheses about their mode of action have been proposed. They can act by complexing with DNA subsequently leading to a DNA-dependent enzymes selective inhibition and/or through transcription direct inhibition (reviewed in Tidwell & Boykin 2003 and Wilson et al. 2005). Evidence indicates that diamidines interfere with kinetoplast function in trypanosomatids through selective association of frequent AT-rich regions in kDNA minicircles, perhaps involving DNA processing enzymes (reviewed in Werbovetz, 2006). Other proposed mechanisms of action against trypanosomatids include inhibition of proteases, topoisomerases, polymerases, protein kinase A, and phospholipid synthesis (reviewed in Soeiro et al., 2005). Furthermore, thermodynamic studies have shown that although some aromatic diamidine analogues, such as reverse amidines or arylimidamides, are weak DNA binders, they are able to induce profound changes in the topology of T. cruzi kDNA and other DNA sequences, making their functionality impossible (Daliry et al., 2011).
In order to overcome the limitations of using aromatic diamides in medical and veterinary practice, the search for new aromatic dications has been extensively investigated, including the prodrugs synthesis, which are converted to aromatic diamides by enzymes that metabolize them (reviewed in Wilson et al., 2005). Parafuramidine (DB289), a furamidine pro-medication (DB75) has undergone clinical trials against human African trypanosomiasis (reviewed in Bouteille et al., 2003 and Soeiro et al., 2005; Mathis et al., 2006). The DB289 pharmacokinetics was investigated in rats and in cynomolgus monkeys (Macaca fascicularis) showing that oral doses were well absorbed and converted to its active metabolite DB75, and also that the prodrug did not exhibit high toxicity, pointing to its potential use for the treatment of parasitic infections (Midgley et al. 2007). Unfortunately, clinical trials using this parafuramidine have been discontinued due to liver and kidney toxicity (Soeiro et al., 2013).
DB75 and its N-phenyl substituted analog DB569 were active on T. cruzi, the analog was more active on different strains and evolutionary parasite stages, with inhibitory values in the low micromolecular range (De Souza et al., 2004). DB569 was also able to increase survival rates and reduce cardiac parasitism by reversing electrocardiographic changes and significantly reducing the CD8+ T cells number of in cardiac inflammation in mice during acute and chronic infections (De Souza et al., 2006a, 2007). DB75 and DB569 induced apoptosis-like cell death in T. cruzi trypomastigotes, with the second compound showing a greater ability to induce this phenomenon (De Souza et al., 2006b). Another strategy for the analogues development is the introduction of structural variations in the diamidines cation centers (Stephens et al., 2001), leading to the reverse amidines synthesis, in which the imino group is attached to the aniline-type nitrogen rather than directly to the aryl ring. These compounds have been shown to be active on T. cruzi and L. donovani (Stephens et al. 2003). Four reverse amidines, also called arylimidamides, DB702, DB786, DB811 and DB889, and a closely related diguanidine (DB711) showed high activity, in the low micromolar range, on blood trypomastigotes and intracellular amastigotes even after treatment for only 2 h, and the main ultrastructural changes in both forms of the parasite were in the nucleus and mitochondria (Silva et al. 2007a,b).
Regarding the new arylimidamides analysis (AIAs,) preclinical trials with different T. cruzi strains in vitro and in vivodemonstrated the promising potency of m-terphenyl bis-AIA 35DAP073, which, in comparison with Bz, was shown to be 26X and 100X more effective on trypomastigotes (strain Y) and intracellular amastigotes (Tulahuen strain), respectively. This AIA was also active on the Colombian strain. The trypanocidal effect was not associated with the biogenesis of lyid bodies in the host cell, as investigated by oil red staining. Both active (35DAP073) and inactive (26SMB060) AIAs showed similar activation profiles. Due to the high SI values, two AIAS (35DAP073 and 35DAP081) were selected for in vivo analysis, but the acute toxicity assay led to the 35DAP081 exclusion. Assays with 35DAP073 mice infected with strain Y in the acute phase, revealed that treatment for two days led to a 46-96% reduction in parasitemia. But ten daily doses using the Colombian strain resulted in toxicity to the animals, preventing treatment for longer periods. Combination 35DAP073 (0.5 mg/kg) therapy with Bz (100 mg/kg) for 10 days resulted in greater parasitemia suppression and 100% the animals survival. Quantitative PCR (qPCR) showed a considerable reduction in parasite load when compared to either Bz or AIA monotherapy (Guedes-da-Silva et al., 2016).
Nitrocompounds are generally avoided in medicinal chemistry programs due to the nitro grouping presence that creates concern about toxicity issues associated with DNA alterations, but at the same time, this functional grouping is often related to desired biological activity (Walsh & Miwa, 2011; Patterson & Wyllie, 2014; Keenan & Chaplin, 2015; Francisco et al., 2016). Regarding Chagas disease, fexinidazole (Fig. 5a) has been evaluated in murine models of acute and chronic infection, leading to high cure rates and reduced myocarditis (Bahia et al., 2012, 2014a; Caldas et al., 2014). Using the bioluminescence technique, in animals infected with the T. cruzi CL Brener strain (CL Brener) (Lewis et al., 2014) it was observed that fexinidazole and fexinidazole sulfone (Fig. 5b) proved more effective than Bz and Nif, considering animals with negative bioluminescence cured after immunosuppression with cyclophosphamide (Francisco et al., 2016). A phase II clinical trial was conducted in Bolivia (NCT02498782) with chronically ill patients, but after recruitment of 47 participants, questions regarding safety and tolerability arose, and it was decided to end the trial without new patients inclusion, and a high efficacy rate was observed (DNDi, 2016; Barreira & Blum, 2018). A new proof-of-concentration study has been designed and will be developed at 4 sites in Spain with patient recruitment planned for 2017 (DNDi, 2017).
3-Nitro-1H-1,2,4-triazoles from aromatic and aliphatic amine groups, piperazines, piperazides, amines and sulfonamidase were assayed by Papadopoulou and collaborators. Many of these 3-nitrotriazoles showed activity on intracellular amastigotes at the nanomolar level and with SI > 200, while the nitro group removal led to inactive compounds. The 3-nitrotriazoles trypanocidal activity has been associated in part with a NTR-I activation (Papadopoulou et al., 2011, 2012, 2013a,b, 2014, 2015a-c, 2016a, 2017a). Some of these compounds were designed as bifunctional agents aiming to be NTR-I substrates and reversible CYP51 inhibitors showed high activity in vitro and in vivo on T. cruzi (Papadopoulou et al., 2015a,b). By live imagingtechnique, the 3-nitrotriazoles that showed the highest activity were an amide (Fig. 6a) and a thiophene sulfonamide (Fig. 6b) leading to no signal from the parasite (Papadopoulou et al., 2013b). In another murine model infected with strain Y, five compounds were highlighted this thiophene sulfonamide (NTR-I substrate but not CYP51 inhibitor), one amide (Fig. 6c) (NTR-I substrate and weak CYP51 inhibitor) and three aryloxyphenylamides (Fig. 6d-f) (NTR-I substrates and CYP51 inhibitors) with high in vitro activity and low toxicity to the host cell (Papadopoulou et al, 2013b, 2014, 2015a,b) were tested using a long treatment time. These 3-nitrotyrools led to parasitemia suppression, 100% survival, and no myocardial inflammation in a fewer number of doses than Bz (Papadopoulou et al., 2017b).
4. Conclusions & challenges
The new drugs development requires the disciplines interaction of molecular and cellular biology, chemistry, biochemistry, pharmacology, and toxicology. The genomics advent, rapid DNA sequencing, bioinformatics, and automated high-throughput screening has strengthened the interaction between different expertises groups in order to search for compounds with high efficacy, including those that can be administered to immunosuppressed patients, with no or low toxicity and short production times.
Regarding the lack of translation between preclinical and clinical trials, the literature clearly points to the need for more feasible and standardized protocols in animal models. A recent study was conducted on treatment with Bz and with VNI, a CYP51A potent inhibitor, in mouse models using both sexes, different parasite strains and therapeutic regimens (Guedes-da-Silva et al., 2016). The results obtained corroborated previous data from the literature showing that females are less vulnerable to infection than males in treatment with both Bz and VNI, suggesting that models with male animals are more suitable. Furthermore, zi preventive protocols (compounds administered 1 day post-infection) may result in unreliable treatment success as the infection would not be spread in different tissues and organs, and thus such protocols should be avoided in vivo screenings. Another consideration is the immunosuppression methods relevance in order to verify the new compounds therapeutic profile, in addition to the molecular diagnostic techniques usefulness (quantitative PCR) to verify efficacy in experimental animals (Guedes-da-Silva et al., 2016).
In a literature review in 2002, we mentioned that “…. An ideal drug does not yet exist and it will possibly be a long time before to be obtained” (Coura & De Castro, 2002). As cited by Moraes & Franco in 2016, millions of compounds have been assayed in HTS campaigns. Unfortunately, despite great advances in technology and knowledge about T. cruzi and the infection development, the picture remains bleak, and in the near future, we see three promising avenues: new regimens of old drugs that will reduce doses and treatment time, the combinations of Bz or Nif use (such as with CYP51 inhibitors), as is the study priority under development by DNDi (DNDi, 2015; Barreira et al., 2016; Barreira & Blum, 2018), and drug repositioning. As reported by Araújo-Lima et al. (2018), the statins activity and selectivity such as atorvastatin (AVA) on T. cruzi in vitro models of combination therapy with Bz showed a synergistic interaction between the two drugs on intracellular trypomastgote and amastigote forms. Also, another recent study, using fixed ratio protocol of Bz with metronidazole (Mtz), a commercial nitroimidazole with a broad antimicrobial spectrum and a reasonable safety profile, showed in vivo that such a combination (Bz 10+Mtz 250) prevented 70% of animal mortality, in addition to protecting from electrical cardiac changes triggered by (Simões-Silva et al., 2017). Taken together, these studies emphasize the importance of drug repositioning and combination treatment for alternative therapies for this neglected and silent pathology.
José Rodrigues Coura
Laboratory for Parasite-caused Diseases, Oswaldo Cruz Institute/Fiocruz
Joaquim Romeu Cançado
Federal University of Minas Gerais and Carlos Chagas Foundation
The Chagas disease specific therapeutics history can be divided into three periods: (i) from the disease discovery in April 1909 until the Carlos Chagas death in November 1934, and soon after in 1935 the “Manual of Tropical and Infectious Diseases” release by Evandro Chagas, on behalf of his father and himself as authors, where they say: “Trypanosomicidal drugs have been tried without any success”; (ii) from 1936 to 1960, when numerous drugs were empirically tried with controversial results; and (iii) from 1961, when Zigman Brener clearly demonstrated the nitrofurazone activity in schemes of prolonged duration in curing experimental mice infection by Trypanosoma cruzi.
The chapter on American Trypanosomiasis in the Manual of Tropical and Infectious Diseases by Carlos Chagas and Evandro Chagas, published in 1935, in its 36 pages, dedicates only one paragraph with six lines to the treatment topic, where it says: “There is no specific treatment for American Trypanosomiasis until the present time. Drugs with trypanomicidal actions have been tried by numerous researchers without any success. Some clinical syndromes may experience symptomatic therapeutic action, performed according to their manifestations and evolution”. Although they speak “trypanosomicidal drugs have been tried by numerous researchers” they do not cite references to published work on the mentioned drugs. Possibly because the results were negative, the researchers, as usual at the time, did not want to publish them.
In an excellent review by Brener in 1968 in the chapter on “Experimental therapeutics of Chagas disease“, in the most complete work on the disease published until then, the book on Chagas disease, organized by Cançado, with the collaboration of the greatest Brazilian specialists on the disease at that time, only two works had been published on experimental therapeutics of Chagas disease until 1935, the work by Mayer and Rocha Lima in 1912 on Atoxil (arsemical), fuchsin (a rosaniline dye) and emetic tartar (pentavalent antinomial) and by the same authors in 1914 on mercuric chloride, both without favorable results. In the thorough 1968 review Brener mentions 23 other chemotherapeutics and over 30 antibiotics used from 1936 to 1962, of which only the following had any suppressive effect on Trypanosoma cruzi infection: “Bayer 7602” bisquinaldine, phenanthridines, 8-aminoquinoleines, trivalent arsenicals (Bayer 9736) and Spyrotrypan, “acromycin” or “stilomcin”, nitrofurans and “Flagyl” (acetamide-5nitrothiazol Imidazole) and particularly the nitrofurans which we will discuss later.
In an therapeutic attempts of Chagas disease analysis in the 50 years since its discovery (1909-1959), Cançado, in his book published in 1968, says “the literature review on the Chagas disease treatment of immediately reveals extreme poverty” and then presents the following numbers that confirm his assertion: of 96 papers presented at the International Congress on Chagas disease, held in Rio de Janeiro in July 1959, only four refer to the disease therapeutics and of these only one to clinical therapeutics, and adds, and adds that, of the 1,369 papers on Chagas disease identified by the Brazilian Institute of Bibliography and Documentation (IBBD, acronym in portuguese) in the same period (1909-1959), there were only 69 on treatment, of which 43 were experimental therapeutics and 26 clinical therapeutics. Finally, he mentions the fact that has always impressed all of us who work with Chagas disease: none of the 64 works by Carlos Chagas cited by IBBD (acronym in portuguese) is dedicated to the disease treatment. Certainly Carlos Chagas and/or his collaborators must have conducted therapeutic experiments for the disease, particularly with arsenicals and antimonials, used respectively since 1906 for syphilis (Paul Enrlich) and 1912 for leishmaniasis (Gaspar Vianna).
Still in the chapter on Chagas disease treatment, Cançado makes a literature critical analysis, showing the methodological fragility of the works on therapeutics great majority. On the one hand because most of them are in the acute phase and using the remission of symptoms and clinical signs and parasitemia as parameters, which usually occurs in this phase even in untreated patients, and on the other hand because of the lack of systematization in the cure and the definitive proof control that would be the serology negation, revealed by the complement fixation, a diagnostic method of the time, most often not applied or with inconsistent results.
Among the chemotherapeutic agents used from 1936 to 1960 in an attempt to treat Chagas disease are the quinoline derivatives and several other antimalarials, arsenobenzoles and other arsem radicals, phenanthridines, gold, bismuth, copper and zinc salts, sodium iodide, gentian violet, aminopterins, para-aminosalicylic acid, nicotinic acid hydrazide, sulfonamides, antihistamines, ACTH and cortisone, stilomycin derivatives, amphotericin B, and more than 30 antibiotics, and some nitrofurans with negative or questionable results, as reviewed by Coura e Silva, and by Brener and Cançado.
Starting in 1961, when Brener, reviewing the nitrofurans experimentally studied by Packchanian in experimental T. cruziinfection, clearly and undoubtedly demonstrated that nitrofurazone (5-nitro-2-furaldehyde-semicarbazone) in a scheme of prolonged duration (53 days on average) at a dose of 100 mg/Kg/day cured more than 95% of chronically infected mice, a new era in the Chagas disease treatment was opened. Soon after, Ferreira and collaborators also treated 10 disease acute cases with nitrofurazone, “with good results and few side effects,” but then found that five of them again tested positive for xenodiagnosis. Coura and collaborators report in papers published in 1961 and 1962 that they treated 14 chronic cases with nitrofurazone (Furacin) in progressive doses of 10 mg/Kg/day in the first week, 20 mg/Kg/day in the second week, and 30 mg/Kg/day in the third week; the side effects in the first four cases were so intense (sensitive polyneuropathy) that the treatment had to be stopped. They restarted treatment with 10 mg/Kg/day in 10 other patients, with 5 tolerating treatment despite side effects (anorexia, weight loss, paresthesias, and sensory polyneuropathy) for 60 days; one of these patients with recent chronic infection (18 months) tolerated treatment with 20 mg/Kg/day for 53 days. Of the six patients treated for a prolonged period (more than 50 days), two were considered cured, based on the xenodiagnostic persistent negativity and complement fixation reaction (Guerreiro and Machado reaction), which never became positive again. Cançado and collaborators in 1964 also treated five chronic patients with nitrofurazome at a dose of 10 mg/Kg/day over varying periods with the tolerance ranging from 10 to 34 days, with therapeutic failure. The final conclusion was that nitrofurazome could be curative but patients could not tolerate the side effects at the doses and time required for cure.
In the late 1960s and early 1970s two new drugs emerged with better prospects for the treatment of Chagas disease both for their curative potential, particularly for the acute phase, and for their tolerance: nifurtimox a nitrofuran: 3-methyl-4-(5′-nitrofurfurylidenoamino)tetrahydro-4H-1, 4-thiazin-1,1-dioxide (Bayer 2502) marketed as Lampit and the benznidazole (N-benzyl-2-nitroimidazole acetamide (RO 7-1051) marketed as Rochagan® in Brazil and Radanil® in Argentina. Nifurtimox developed by Bock and collaborators and benznidazole developed by Richle have proven active in vitro and in vivo against T. cruzi. The nifurtimox production since the 1980s has been discontinued, initially in Brazil and later in Argentina, Chile, and Uruguay, and benznidazole in Brazil is being passed on by Roche to Pernambuco State Pharmaceutical Laboratory (Lafepe, acronym in portuguese). The pharmaceutical industry disinterest in the production of drugs for Chagas disease, considered as “social drugs“, is linked to the low demand, restricted only to some countries in Latin America, for low-income populations and, therefore, with small profit margin.
Nifurtimox and benznidazole have been widely used by several researchers, especially in Brazil, Chile, and Argentina, including Cançado and collaborators, Cançado and Brener, Bocca-Tourres, Rubio and Donoso, Schenone and collaborators, Rassi and Ferreira, Rassi and Luquetti, Coura and collaborators, Macedo and Silveira, Viotti and collaborators, Andrade and collaborators, Sosa Estani and collaborators, Cançado, and Lacunza and collaborators, among several others who have published their results.
The results obtained with both drugs varied according to the disease stage, the treatment duration, the patients age, and the geographical area of their origin. The best results have been obtained in the disease acute phase, in children and patients with recent infection, using nifurtimox at a dose of 8 to 10 mg/Kg/day or benznidazole 5 to 7.5 mg/Kg/day for 60 to 90 days. In the chronic phase and in adult patients the best results were obtained in southern Brazil, Argentina and Chile, therefore in the Southern Cone, probably due to the type of T. cruzi strain in this region. In summary, it can be said that the cure percentage was 60 to 80% in the acute phase and 10 to 20% in the chronic phase, according to the various authors and geographical areas. Some authors who have obtained “high cure percentages” in the chronic phase have relied on negative xenodiagnostic or PCR, which can be negative in more than 50% of patients with reduced parasitemia, even if not cured.
The most frequent side effects with nifurtimox were anorexia, weight loss, psychological excitability or drowsiness, and digestive manifestations such as nausea, vomiting, and occasionally intestinal colic. The side effects with benznidazole can be classified into three types: (i) hypersensitivity manifestations such as dermatitis with rash (usually appearing between day 7 and 10 of treatment), peri-orbital or generalized edema, fever, lymphadenopathy, and muscle and joint pain; (ii) bone marrow depression, among which neutropenia, agranulocytosis, and thrombocytopemic purpura; (iii) peripheral polyneuropathy represented by paresthesias and polyneuritis.
Although nifurtimox and benznidazole have been a breakthrough for the Chagas disease treatment, they are far from being considered ideal drugs. The ideal drug for the Chagas disease treatment should meet the following requirements: (i) produce a parasitological cure in acute and chronic cases and avoid the disease evolution, being effective with few doses in a short term (10 to 15 days); (ii) not produce important side effects or teratogenicity and not induce parasite resistance; (iii) be cheap, easy to apply and accessible to patients.
From the 1990s on, facing the nifurtimox and benznidazole limitations, some drugs such as allopurinol, an analogue of hypoxanthine, a xanthine oxidase inhibitor and purine synthesis, used for the gout treatment, and azole antifungals such as ketoconazole, fluconazole and itraconazole, which showed some in vitro activity against T. cruzi, were used in experimental animals and in man with controversial results, as published by Lauria-Pires and collaborators, Galerano and collaborators, Brener and collaborators, de Castro, Apt and collaborators, and Molina and collaborators.
The serious problem that leads to controversies and a huge waste of time is the lack of criteria in clinical and experimental in vivo evaluation regarding partial results obtained in vitro culture. It is clear that this is a necessary and indispensable first step, but it must be followed with a rigorous study in an experimental model in animals, with different strains of T. cruzi, before any announcement and experimentation in humans, which, in addition to toxicity studies, must be done with extreme scientific rigor before the positive results proclamation, most often false due to lack of criteria in the parasitological cure control and the disease evolution and that must always be done with a control group.
The development of an anti-parasitic drug can arise through experiments with natural or synthetic products that have similarity with compounds with recognized activity for other diseases or through specific metabolic targets for a particular parasite to be targeted, as reviewed by Coura & de Castro. As perspectives for the Chagas disease experimental treatment, several targets are being opened through T. cruzi metabolic and biochemical studies, among which the synthesis of sterols and enzymes essential for this parasite development and multiplication, as reviewed by Do Campo.Considering that ergosterol is the T. cruzi main sterol, in the last decade research has been directed towards the development of an effective inhibitor of this sterol. Urbina and collaborators developed DO870 that cured a high percentage of animals infected with T. cruzi in the short and long term. More recently Molina and collaborators demonstrated that the triazole posoconazole (SCH 56592 Schering-Plouch) inhibits T. cruzi epimastigote proliferation and ergotenol synthesis up to 100 times more than DO870, including from nifurtimox- and benznidazole-resistant strains. Posoconazole is at the moment the great hope in the Chagas disease treatment and is already in the initial phase of experimentation in humans.
Milena B. P. Soares, Bruno Solano de Freitas Souza, Ricardo Ribeiro dos Santos
Gonçalo Moniz Institute/Fiocruz
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The search for methods to repair injuries caused by chronic or traumatic diseases has been driven by the discovery of stem cells with the ability to self-replicate and differentiate into various functional cell types. The demonstration that adult stem cells can act therapeutically by releasing trophic and immunomodulatory factors has also helped to increase the potential therapeutic indications. With regenerative medicine, instead of replacing an injured organ, the possibility of repairing it is glimpsed, and cell therapy is one of its most promising tools. The heart damaged by chronic Chagas disease is a potential target for regenerative medicine, aiming to restore functional cells and organ function.
Chronic Chagas’ disease is still today a disease without effective therapeutic possibilities other than heart transplantation. Because of the low organ harvesting rate, most Chagas disease patients on the transplant waiting list die before receiving a new heart. It is a disease characterized by a myocardium progressive destruction by an inflammatory response that can progress to cardiomegaly and malfunction of the heart, resulting in high morbidity and mortality of patients. Therefore, a therapy that can cause an improvement in cardiac function, and that is accessible to the Chagas’ disease patients population, is of great interest.
Among the various cell types that are being investigated for their potential use in cell therapies for cardiac regeneration are: bone marrow mononuclear cells, mesenchymal stem cells, endothelial precursors, cardiac precursors, and cells differentiated from pluripotent stem cells (embryonic and induced pluripotency stem cells or iPSCs).
Bone marrow was a source of stem cells with immediate application in clinical studies, not only because it is easy to obtain, but mainly because of the accumulated experience with their clinical use for the hematological and oncological diseases treatment, in which they are routinely used in clinical practice. Therefore, the bone marrow cells potential use in regenerative medicine has been intensively studied, and this was the first cell population investigated for the Chagas’ disease treatment.
To test the bone marrow cell therapy efficacy in the Chagas’ cardiomyopathy treatment, the experimental model of isogenic mice chronically infected with the T. cruzi Colombian strain, which causes the chronic Chagas’ cardiomyopathy development, was used. Bone marrow cells were obtained from normal or chronic Chagas’ mice and injected intravenously into mice in the infection chronic phase. Transplanted mice had a decrease in inflammation and fibrosis after transplantation when compared to untreated controls. The bone marrow cell therapy effects were long-lasting, as the inflammatory cells number and the fibrosis area remained reduced up to six months after treatment (Soares et al, 2004).
Using bone marrow cells obtained from mice transgenic for green fluorescent protein (GFP), transgenic cells were found to be present in the myocardium, indicating that some of the injected cells migrate to the heart. Some of these cells showed cardiomyocyte morphology and myosin expression, indicating transplanted cells possible differentiation into this cell type or fusion with cardiomyocytes from the recipient animal. However, these were uncommon findings. In fact, DNA microarray analyses have shown that bone marrow mononuclear cell therapy reverses most of the changes in gene expression altered by T. cruzi infection in mouse hearts, including several factors associated with inflammation and fibrosis (Soares et al, 2010). Therefore, we demonstrate that this cell population has potent immunomodulatory potential, but low cardiac functional cell replacement capacity.
In experimental T. cruzi infection in rats, Guarita et al. (2006) studied the cell therapy effects, which consisted of direct administration into the left ventricular wall of bone marrow-derived mesenchymal cells co-cultured with skeletal myoblasts for 14 days, on the heart’s ejection fraction improvement. The authors observed a significant improvement in ejection fraction after cell therapy in chronically infected animals with an ejection fraction below 37%. A decrease in left ventricular end-systolic and end-diastolic volumes, and the myogenesis and angiogenesis presence after treatment were also observed.
Another cell population that has been tested is mesenchymal stem cells, which are adult cells found in various tissues, including bone marrow and adipose tissue. Treatment of mice with chronic Chagas’ heart disease with mesenchymal cells from human adipose tissue and mouse heart caused a inflammation and fibrosis reduction in the heart (Larocca et al, 2013; Silva et al, 2014). Cell migration analysis showed that mesenchymal stem cells are found in the liver, lungs, and spleen, and few cells migrate to the heart (Jasmine et al, 2014).
In a study carried out at the Santa Izabel Hospital in Salvador, Bahia, Chagas disease patients with NYHA grades III and IV heart failure were selected (Vilas-Boas et al, 2006). From each patient, 50 mL of bone marrow was aspirated by puncturing the iliac crest under local anesthesia. The marrow aspirate was subjected to Ficoll gradient centrifugation to isolate a fraction enriched with mononuclear cells, which was injected slowly into the left and right coronary system on the same isolation day. One month after transplantation, the patients received daily injections of G-CSF for five days to induce bone marrow stem cells mobilization into the peripheral blood, with the goal of inducing a boost in stem cell supply for injury repair. No adverse effects have been observed from bone marrow mononuclear cell transplantation, nor from G-CSF treatment. There were no significant changes in CKMB and troponin levels, muscle damage markers, as well as in the arrhythmias number and intensity, indicating that this is a safe and feasible protocol, as described by Vilas-Boas et al. (2006). Left ventricular ejection fraction analysis indicates improved cardiac function six months after the procedure. Similarly, an increase in walking time in the corridor test and improvement in New York Heart Association functional class were observed. Finally, the patients reported an improved quality of life (Minnesota questionnaire). The patients’ serum sodium levels before treatment were below normal, as is often the case in patients with this heart failure degree.
Biodistribution study through bone marrow mononuclear cell labeling and scintigraphy showed that the transplanted cells are mainly located in the liver and spleen, but are also found in the heart, although in smaller quantities (Barbosa da Fonseca et al, 2011).
To prove whether cell therapy is effective in the treatment of chronic Chagas’ cardiomyopathy, a multicenter, randomized, double-blind study (MiHeart), funded by the Ministry of Health, was developed. In that study, which included 234 chagasic patients, it was shown that intracoronary injection of autologous bone marrow mononuclear cells did not cause improvement in left ventricular function or quality of life (Ribeiro dos Santos et al, 2012).
Although observations in the animal model, as well as in the clinical study, indicate that stem cell therapy has a promising future in the chronic Chagas’ cardiomyopathy treatment, we still have a long way to go in order to determine the best cell type, how many and which doses to use, the application moment, and above all, the mechanisms through which the cell therapy acts. Chronic Chagas’ cardiomyopathy presents peculiarities in relation to other cardiomyopathies, such as intense inflammation, which can facilitate or hinder the stem cells action. Because it is a progressive disease, interventions at earlier stages are likely to be more effective and promote delay or block the disease progression, rather than aiming for function recovery in end-stage heart failure hearts.
In addition to adult stem cells, such as mesenchymal cells, other cells generated from pluripotent stem cells (embryonic or induced pluripotency stem cells – iPSCs) or by direct reprogramming, such as cardiomyocytes and cardiac progenitor cells, should be tested in the preclinical phase to determine safety and potential efficacy, to serve as the basis for future clinical trials. Another question to be solved is how to ensure the survival, engraftment, and electrical and mechanical integration of the transplanted cells into the heart tissue. To achieve this goal, interdisciplinary strategies, through associations of cellular products with nanotechnology, biomaterials, or gene therapy, will be increasingly used.
Bone marrow cell therapy is not intended to treat Chagas disease. It does not interfere with T. cruzi infection, as far as we can tell from the observation that there is no change in the treated animals parasite load or parasitemia. It is also unknown whether this therapy has an effect on the heart-aggressive immune response. This is a reparative therapy for the damage caused during years or even decades of myocardium aggression resulting from Chagas disease. What is expected is that these individuals can live with the parasite without symptoms, as is the case with most individuals infected with T. cruzi in the disease undetermined form.
Laboratory of Interactions Biology, Oswaldo Cruz Institute/Fiocruz
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Andréa Silvestre de Sousa
Oswaldo Cruz Foundation, Evandro Chagas National Institute of Infectious Diseases, Rio de Janeiro, Rio de Janeiro, Brazil.
Federal University of Rio de Janeiro, School of Medicine, Department of Internal Medicine, Rio de Janeiro, Rio de Janeiro, Brazil.
The neglectful character associated with Chagas disease can be evidenced by the low interest of the pharmaceutical industry for research and development of new drugs directed to this infection treatment. The only commercially available drugs with established antiparasitic efficacy are the same ones that have been in use for more than fifty years: benznidazole and nifurtimox. The negligence is also notorious when evaluating the scientific evidence quality and the criteria heterogeneity in the existing guideline recommendations in the various Latin American countries. As a consequence, the obstacles in the standardized conducts application by managers and health professionals are exacerbated. Moreover, losses occur in the promotion and continuing education activities standardization, which are more and more desired in the face of the universities silence regarding neglected tropical diseases (NTDs), thus perpetuating the conditions of those affected vulnerability.
As for existing drugs, nifurtimox, marketed by the German company Bayer, was pioneered in 1965. Despite its proven efficacy, the serious and frequent adverse reactions, including anorexia, weight loss, paresthesias, psychic agitation, nausea, and vomiting, were responsible for the reduced use of this drug, which is now unavailable in many countries, including Brazil. In our country, in specific situations, such as suspected resistance or serious adverse effects associated with benznidazole, nifurtimox can be requested from the Health Surveillance Secretariat (SVS, acronym in portuguese) of the Ministry of Health (MS, acronym in portuguese), which obtains the drug from the Pan-American Health Organization (PAHO).
Benznidazole, a substance from the same pharmacological class as nifurtimox, was first used in the late 1970s. It has similar efficacy, but fewer adverse reactions. Currently, its distribution is carried out by two laboratories: the Brazilian Lafepe (acronym in portuguese) (Pernambuco State Pharmaceutical Laboratory) and the Argentinean Elea, representing the only trypanocide option marketed in our country.
More recently, other azoles, such as posaconazole and ravuconazole, have been tested in the Chagas disease treatment, but the greater efficacy and benznidazole lower cost reduce the chance of using these new drugs in isolation. New therapeutic schemes, with drug combinations and dose changes are being tested. In light of this, benznidazole and nifurtimox persist as the only therapeutic options to date, even though they were first introduced in the 1960s and 1970s.
According to the World Health Organization (WHO), the ideal drug to be used in the Chagas disease treatment would be one capable of promoting parasitological cure in both the acute and chronic phases, with guaranteed efficacy after one or a few doses, low cost, devoid of adverse or teratogenic effects, and unable to induce resistance. Unfortunately, there is, until now, no such utopian substance. On the contrary, our current therapeutic options are far removed from the characteristics mentioned here.
The factors that influence the both drugs cure efficacy are varied: the disease stage evolution at the time therapy is instituted; treatment dose and duration; age and immune status of the treated individual; and patient geographical origin, representing the Trypanosoma cruzi genetic variability and its natural resistance to treatment. Thus, cure rates based on serology and xenodiagnosis can reach 80% in Argentina, Chile, and southern Brazil, but only 40% in Brazilians from the central and southeastern regions of the country.
Similarly, serological negativity can occur in 80% of cases when treating children in the disease acute phase, and less than 20% after treating adults in the chronic phase. Low parasitemia in the later stages would decrease the success chance. On the contrary, there is no doubt about the benefit of treating all disease acute forms, their transmission route regardless, including maternal-fetal cases, laboratory accidents, and reactivation in immunosuppression cases. The disease stage and the individual age influence not only the treatment effectiveness, but also the incidence of adverse events and the disease progression expected rate. In this rationale, children are once again the preferred targets, not only for the greater efficacy described, but also for lower rates of progression and adverse events.
The major limiting factor in discussing therapeutic efficacy in chronic Chagas disease is the cure-defining marker absence. If the serological negativity criterion is established, the observation period for this event can be decades, making it infeasible to schedule clinical trials with serological endpoints. The use of a surrogate parasitological endpoint, such as T. cruzi DNA assessed by negative PCR, does not indicate cure, because negative PCR does not mean infection absence. PCR should only be used as a treatment failure marker if it remains positive after the medication course.
Other surrogate endpoints, such as reduced disease progression, have been used in some studies. Thus, the lowest electrocardiographic progression compared to placebo is described among patients treated with benznidazole, although seroconversion was as low as 15% after 9.8 years of follow-up. Based on observational studies, both compounds are considered effective in reducing the duration and clinical disease severity, clinical high importance outcomes.
The benznidazole recommended dose in most guidelines is 5 mg/kg/day (100mg tablets), divided into two or three daily doses, maintained for 60 days. In this period, adverse reactions must be evaluated, the most frequent being dermatological, with exanthemas, pruritus, and flaking, in addition to anorexia, weight loss, paresthesias, and peripheral polyneuropathy, the symptoms of which may remain for months after the treatment end. More rarely, bone marrow depression, thrombocytopenic purpura, and agranulocytosis may occur. The treatment discontinuation rate associated with adverse reactions can range from 25 to 41%.
Recently, a strategy that sets the maximum daily dose at 300mg has been adopted, extending the treatment time, in days, according to the individual’s weight (e.g., for an individual weighing 80Kg, the dose of 300mg/day should be maintained for 80 days). This conduct, adopted in the BENEFIT clinical trial, made the discontinuation rate predicted by the sample calculation (17%) lower than that actually found (13.4%). In the same study, the assessed adverse effects incidence (23.9%) was lower than predicted, based on literature data.
In the disease acute phase, the understanding of the imperative need for etiologic treatment is universal, transmission route regardless. However, doubts regarding the benefit persist for the chronic phase. Unfortunately, the autoimmunity theory that emerged in the 1980s may have mistakenly put specific treatment in the background for chronic cases. The pathogenesis current understanding, especially with the advances in molecular biology and reactivation recognized cases in immunosuppressed patients, leaves no doubt of the parasite importance in the disease evolutionary process in its chronic form. The etiological treatment, therefore, has its theoretical basis anchored in reducing the stimulus to the inflammatory process and controlling disease progression, with positive responses in observational studies and experimental models.
Although it is rational to use trypanocide therapy to control the disease morbidity, its efficacy in the chronic form is considered uncertain in individuals with a longer duration of the disease. Thus, even though the current Brazilian consensus has expanded its treatment recommendations, doubts persist among researchers, ratified in this document, regarding the benznidazole use among individuals over 50 years old and among those with the symptomatic chronic form, especially in chronic Chagas’ cardiopathy.
In general, especially for doubtful situations, it is recommended that the decision about treatment be shared between doctors and the infected individuals, scoring risks associated with adverse effects and potential benefits. Under 50 years old, achieving the no progression benefit to heart disease offsets most of the risks.
Importantly, childbearing age women treatment is a high impact, low cost strategy in reducing maternal-fetal transmission. Pregnant women should not receive trypanocide therapy because of the teratogenicity potential risk, except in severe acute cases where maternal life is prioritized.
It is pointed out that most individuals over 50 years old must have been infected more than 30 years ago. If at diagnosis they are still in the undetermined chronic form, they are unlikely to progress to severe cardiac forms. In these cases, because the benefits are less clear and the risks are possible, routine treatment is not recommended, although the decision should be discussed between the parties involved. However, if the comorbidities are small, life expectancy is high, and above all, if the infection supposedly occurred in adulthood, one tends more easily to opt for etiological treatment. This last point receives special importance in view of the recent change in the Chagas disease epidemiological profile in Brazil, where most of the acute cases occur in the Amazon, among individuals of all age groups. In this scenario, age cannot be used to infer the disease time course as in classical vector transmission. Thus, in these particular cases, the age limit for etiological treatment should not be considered for the treatment indication.
As for the symptomatic chronic forms, patients in the digestive form should be managed as for the indeterminate chronic form, with guidance to use specific treatment until 50 years of age, except for isolated cases of increased difficulty swallowing and erratic absorption associated with advanced megaesophagus.
The big discussion falls to patients with chronic Chagas’ cardiopathy. There is consensus among all the guidelines that no specific treatment should be performed in advanced heart disease cases. In mild to moderate heart disease, however, despite the BENEFIT study results, a randomized, placebo-controlled clinical trial that evaluated a significant number of patients in Latin America, which showed no benefit in the death primary outcome and associated cardiovascular events, there are still questions about the treatment orientation in these cases. Subgroup and post hoc analyses attempt to justify a consensus therapy guideline among physicians and individuals involved, at least in the Brazilian population, the only one that would achieve a trend toward therapeutic benefit in further analyses. It is curious that, when looking at the T. cruzi genetic variability, Brazil would correspond exactly to the geographical area where the lowest success rates associated with trypanocide therapy historically occur, when compared to the Argentine centers.
An interesting analysis not yet evaluated would be to compare the quality of symptomatic treatment performed in parallel by all centers involved in the BENEFIT study. As the symptomatic treatment benefits of heart disease are undoubtedly for the mortality and other clinical outcomes reduction, these can never be forgotten or put on the back burner, and should be unanimously included in all agendas and demands for the adequate treatment of individuals with chronic Chagas’ disease.
The ideal drug for the Chagas disease treatment, as recommended by the WHO, is not yet part of our current reality. Rather than looking for a magic pill, we should strive for plausible actions, with guaranteed clinical applicability, and relevant outcomes.
In the chronic phase, an extremely relevant conduct would be to promote the treatment of all women with positive serology at childbearing age. This simple, low-cost action extends the disease control by reducing potential new cases arising from maternal-fetal transmission and by limiting morbidity in this younger population segment.
In the undetermined chronic form, it is also relevant to promote the younger individuals treatment, up to 50 years of age, with latent risk of progression to severe symptoms. It should be considered, however, that this arbitrary age limit should not be applied to those infected in adulthood, a condition in which treatment could be more effective, given the disease progression shorter time.For heart disease patients, however, recognizing that definitive structural lesions associated with extensive fibrosis may have already occurred, and in the face of existing scientific evidence that has not demonstrated a clear benefit from trypanocide therapy, one should seek, if not a cure with its questionable criteria, a reduction in morbidity and mortality, and a better quality of life. All this is feasible through symptomatic treatment and cardiovascular rehabilitation, options that are often available, yet poorly applied or relegated to the second line.
J. Antonio Marin-Neto, Anis Rassi Jr, Anis Rassi
Cardiology Division, Internal Medicine Department, University of São Paulo at Ribeirão Preto Medical School, Ribeirão Preto, SP, Brazil (J. Antonio Marin-Neto) and Cardiology Division, Anis Rassi Heart Hospital, Goiânia, GO, Brazil(Anis Rassi Jr, and Anis Rassi).
Abstract: In recent decades the relationship between parasite persistence and the onset and chronic cardiomyopathy progression in Chagas disease has been well demonstrated. In this context, besides the formal indication for antiparasitic treatment in the infection acute phase, patients with the indeterminate form and even those already diagnosed with cardiomyopathy, provided it is not advanced, should be considered for trypanosomicide treatment, based on an individualized decision that should be shared between the physician and the patient himself.
Keywords: Chronic Cardiomyopathy in Chagas disease – Etiologic Treatment – Benzonidazole – BENEFIT Study
In contrast to the consensus recommendation that all patients in the Chagas disease acute phase, regardless of the transmission mechanism, should receive trypanosomicide therapy, the indications for etiologic treatment in the chronic phase remain debatable. The elucidation of the etiologic treatment role in the Chagas disease chronic phase has been hindered by several factors[i]: first, the misconception that autoimmunity would be the main pathogenetic mechanism in the disease chronic phase; second, the fact that since the benzonidazole and nifurtimox introduction some 40 years ago-the only antiparasitic drugs with proven efficacy-the etiologic treatment has not experienced advances and the emergence of new, less toxic and more effective drugs has not been possible; third, there was the physicians fearful reluctance about undesirable side effects caused by trypanosomicides, especially in patients with manifest cardiomyopathy, coupled with the notion that etiologic treatment in this context would already be late and without effect[ii]
Recently, however, with the better understanding of the Chagas disease natural history, this negative scenario has changed, providing a more rational approach to the various aspects related to the Chagas disease etiological treatment. Thus, although it is now recognized that three other mechanisms are involved in the complex pathogenesis of chronic Chagas disease cardiomyopathy (CCDC) – peripheral autonomic dysfunction, coronary microvascular alterations, and myocardial lesions of an immunological nature – many investigators now define that tissue parasite persistence constitutes the essential factor responsible for the installation and progression of the typical chronic myocarditis that causes reparative and reactive myocytolysis and fibrosis. This concept has evoked renewed interest in antiparasitic therapeutic perspectivesass by rescuing the crucial notion that Chagas disease in its chronic phase remains an essentially infectious nosological entity, during which the human organism, despite its proteiform defenses, cannot completely debilitate the aggressor parasite[iii], [iv].
There is evidence that the inflammatory process intensity correlates markedly with tissue parasite load in chronic T. cruzi[v]infection experimental models. In addition, it has also been shown that the trypanosomicidal treatment with benzonidazole, nifurtimox and fexinidazole, despite not eradicating the parasite reduces the infectious load and, relevantly, causes marked attenuation of myocarditis in these experimental models [vi], [vii], [viii].
Many patients suffer lifelong indeterminate chronic Chagas disease. One of the most burning and intriguing unanswered questions about the disease pathogenesis is the enigma of why only 30-50% of patients infected with T. cruzi develop the chronic clinical forms – especially the most severe and frequent, CCDC – while the others are spared these complications?1Although the individual course of a patient with the Chagas disease indeterminate form is unpredictable, it is now entirely plausible to assume that it represents the clearest opportunity for CCDC spectrum secondary prevention in T. cruzi infected humans[ix]. That is why the most recent Brazilian Consensus on Chagas disease recommends as class I etiologic treatment with benzonidazole for all children under 12 years, and as class IIa for every adolescent < 18 years, and, on an individualized basis, for most adults up to 50 years, cursing with the indeterminate form[x].
The level B evidence supporting these recommendations derives from the few small randomized trials and a few other, observational studies, performed in asymptomatic children and adults with normal ECG at rest[xi], [xii], [xiii], [xiv], [xv], [xvi], [xvii],. The etiologic treatment benefit in such studies could be ascertained only for surrogate outcomes of the clinically most relevant ones, i.e., by indirect evidence of reduced parasite load and ECG changes incidence detected during relatively short follow-up periods compared to the very long natural disease history. This finding stems from the inherently disease very prolonged indeterminate form (i.e., before clinical manifestations appear), preventing the effects on the most clinically relevant outcomes (e.g., death, heart failure, severe arrhythmias) from being investigated in these studies. These researches results were collated into respective systematic reviews and meta-analyses[xviii], [xix], [xx], [xxi].
Until the publication of the BENEFIT study (BENznidazole Evaluation For Interrupting Trypanosomiasis) [xxii], [xxiii] the evidence supporting this indication was based on the aforementioned studies, in particular by the results of meta-analyses comprising three small randomized trials and six observational studies; This meta-analysis results showed that patients treated with benzonidazole had a lower risk of developing clinical outcomes compared to control patients receiving placebo or no treatment (CR, 0. 29; 95% CI, 0.16-0.53)21
This study, the largest in patients with CCDC, enrolled 2854 patients in 49 centers in 5 Latin American countries – 1358 patients in Brazil, 559 in Argentina, 502 in Colombia, 357 in Bolivia, and 78 in El Salvador. Patients were randomly assigned to groups receiving placebo or benzonidazole (5 mg/ kg body weight per day) for 40-80 days, and were followed for an average of 5.4 years23.
Benzonidazole treatment had no significant effect on the study’s primary composite endpoint, which was 27.5% in this group versus 29.1% in the placebo group (RR) 0.93 (95% CI 0.81-1.07; p=0.31).
Although 23.9% of patients in the benzonidazole group transiently discontinued treatment for adverse side events (vs 9.5% in the placebo group, p<0.001), only 13.4% discontinued treatment permanently (vs 3.6% in the placebo group, p<0.001), characterizing lower incidence of these study safety outcomes compared to previous reports for treatment with benzonidazole23.
These results limitations and plausible post-hoc analyses
A CCDC pathogenesis review was recently published, along with a careful post-hoc analysis of the BENEFIT study results and of numerous methodological aspects that may have negatively influenced the investigation scope, with relevant developments to be considered in order to guide medical conduct in the context1.