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www.expert-reviews.com ISSN 1478-7210 2011 Expert Reviews Ltd10.1586/ERI.11.31

Chagas disease, also known as American try-panosomiasis, is a medical and social problemin Latin America. The disease is caused by thehemoagellate parasiteTrypanosoma cruzi [1].The parasite strains are genetically heterogeneousand they have been grouped in six phylogeneticlineages with differential drug resistances andimmune pathogeneses[2].

At the end of the 1980s, the disease affectedapproximately 18 million people and 100 mil-lion were at risk for acquiring the infection in18 countries [3] . Systematic programs aimed ateradicating the domiciliary vector (Triatomainfestans ) and screening blood before transfusionshave led to a decline in the incidence of disease inrecent years and some countries have declared theinterruption of transmission, including Uruguay(1997), Chile (1999) and Brazil (2006)[3,201] .The number of annual deaths from Chagas dis-ease fell from 45,000 in 1985 to 12,500 in 2006(T ABLE 1) [4,201] . The strategy for control of Chagasdisease was based on the detection and treatmentof congenital transmission and on the treatmentof infected children and acute cases.

Currently, however, approximately 28 millionpeople are still at risk of acquiring Chagas dis-ease, with a prevalence of disease at 7.7 million

cases in 15 endemic countries of Latin America(T ABLE 1) [5,201] . These facts demonstrate the neces-sity to maintain and extend the measures fordisease control in order to avoid the establish-ment of other parasitic species in the ecologicalniche left by the domiciliaryT. infestans . The

WHO estimated that mortality among endemicvector-borne diseases in America is higher forChagas disease than malaria, schistosomiasis,dengue, onchocerciasis, lymphatic filariasisand leishmaniasis ( F IGURE 1) . Most deaths occurin a population that is still in employment, sothat the disease causes a signicant economicimpact [3,201] . Epidemiological data may also beunder-reported in most affected countries owingto the absence of a systematic registry of cases.

Natural history of Chagas diseaseT. cruzi infection frequently occurs after themetacyclic trypomastigotes present in vectorfeces invade a human hosts skin. It is estimatedthat the initial phase of disease lasts for approxi-mately 2 months and is asymptomatic in 90%of cases, but patients may present fever, lymph-adenopathy, hepatosplenomegaly, myocarditisand cardiomegaly[6] . Decades after infection,approximately 70% of patients do not present

Paulo MM Guedes 1,Grace K Silva 2,Fredy RS Gutierrez 3 and Joo S Silva 21Department of Microbiology andParasitology, Bioscience Center,Federal University of Rio Grande do

Norte, Natal, Rio Grande do Norte,Brazil2Department of Biochemistry andImmunology, Medicine School atRibeiro Preto, University of So Paulo,Ribeiro Preto, So Paulo, Brazil

3School of Medicine, University Antonio Nario, Bogot, Colombia Author for correspondence:Tel.: +55 163 602 3234Fax: +55 163 602 4590

[email protected]

Chagas disease affects 7.7 million people and 28 million people are at risk of acquiring thedisease in 15 endemic countries of Latin America. Benznidazole and nifurtimox are drugs thathave been used to treat the disease. However, both drugs induce severe side effects. Treatmentwith benznidazole has been recommended for the acute phase (04 months after infection),recent chronic phase (children 014 years of age, treated 4 months after infection) and congenitalinfection. Average cure rates for Chagas disease patients obtained from clinical trials were 97.9%(congenital infection, treatment performed 0 6 months of age), 71.5% (acute phase), 57.6%(recent chronic phase, children 013 years of age) and 5.9% (late chronic phase, great majorityof patients between 15 and 69 years of age). Clinical evidence about the capacity of antiparasitictreatment to avoid, stop or revert heart pathology in indeterminate and cardiac chronic patientsis contradictory. The investigation of novel therapeutic strategies against Chagas disease remainsa priority in the research of tropical diseases. Unfortunately, Chagas disease remains neglectedin the formulation of strategies toward control of this disease. This article focuses on currenttherapeutic approaches to Chagas disease.

K EYWORDS : azoles benznidazole cardiac inammation Chagas disease imidazoles myocarditis parasite persistence Trypanosoma cruzi

Current status of Chagasdisease chemotherapyExpert Rev. Anti Infect. Ther. 9(5), 609620 (2011)


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any clinical signs, characterizing the indeterminate form, whileapproximately 30% develop the digestive and/or cardiac (chronicchagasic cardiomyopathy) forms of the disease[7] . The beginningof cardiac disease is characterized by myocarditis (which includes

the presence of macrophages and CD4+

and CD8+

T lympho-cytes), followed by myocardial remodeling, which contributes tocardiac hypertrophy, brosis, arrhythmias and eventually heartfailure [8] . The digestive form is characterized by intramuralnervous system lesions, tissue parasitism and immune systemaction in the esophagus and colon, which leads to dysfunction(mega-esophagus or mega-colon)[9] .

Chagas disease pathology: autoimmunity& parasite persistenceThe mechanisms of pathogenesis during chronic Chagas diseaseare not completely understood. Evidence of a strong inammatory

reaction and the absence of the parasite in blood or cardiac tissuesby immunostaining methods and the demonstration of antigencross-reactivity due to peptide mimicry have led to the proposalthat autoimmunity is a central factor for cardiac compromiseduring this infection. However, studies using immunosuppressantdrugs also fa iled to ameliorate the disease in chronically infectedsubjects and the induction of tolerance to antigens supposedlyinvolved in the cardiac inammatory reaction did not amelio-rate the grade of myocardial impairment after infection[10] . Inaddition, the development of more sensitive methods demon-strated that the parasite is able to persist within the myocardiumand other tissues for extended periods of time. In fact, there isa correlation between parasite burden (demonstrated either by

antigens or DNA traces) and the intensity of myocardial inam-mation [1114] . This has also been demonstrated in the digestiveform of Chagas disease[15,16] .

It has been discussed whether autoimmune mechanismsparticipate in the pathogenesis of chronic Chagas disease.Bystander activation and molecular mimicry could generateauto-antibodies and autoreactive T cells[1720] . In accordance,

some studies demonstrated the existenceof auto-antibodies against skeletal, ner-vous and cardiac muscle antigens[18] .

Auto-ant ibodies were described againstcardiac myosin, ribosomal P protein,queratin, b -adrenergic and muscarinicreceptors, microtubules and cytoskele-ton-related proteins [2125] . CD4 + T lym-phocytes against cardiac self-antigenshave also been observed in experimentalChagas disease; these cells fromT. cruzi-infected mice recognize cardiac myosin[26] . Passive transfer of CD4+ T cellsfrom mice during chronic infection touninfected mice causes myocardial tissueinammation [27] .

The presence of parasite is importantto initiate and maintain the pathogenicprocess, which may be amplied by auto-immune mechanisms that depend on hostgenetic predisposition, as it is not possibleto make a correlation between inamma-tion and parasite amount in the lesion[1114,28,29] . Because the immunopathologicalprocess continues during the chronic phaseof infection and the parasite is an impor-tant element in cardiac destruction duringhuman Chagas chronic cardiomyopathy[12,30,31] , an efcient etiologic treatmentmay avoid cardiac and digestive derange-ments. Furthermore, in some cases, when

Table 1. Changes in epidemiological parameters ofChagas disease in Latin America.

Factor Year

1990 2000 2006 Prevalence (million) 18 13 7.7

Annual incidence 800,000 200,000 41,200

Population at risk (million) 100 40 28

Annual deaths 45,000 21,000 12,500

Morbidity 700,000 300,000Data taken from [35,114116,201] .

0

1.0

2.0

1.5

0.5

2.5

14.5

14.0

13.5

13.0

12.5

12.0

2002 20152005

Year

E s

t i m a

t e d n u m

b e r o

f d e a

t h s

( t h o u s a n d s

)

Chagas disease

OnchocercosisLymphatic filariasis

LeishmaniasisSchistosomiasis

DengueMalaria

Figure 1. Mortality from endemic disease with vectorial transmission in theAmericas and future projections based on WHO Statistical Information System. Modied by OPAS, 2007. WHO. Global Burden of Disease Estimates. Projections ofmortality and burden of disease to 2030.Adapted with permission from [201] .

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benznidazole was administered and the patient was considereduncured, there was stabilization or reduction of electrocardio-graphic alterations compared with the untreated control group[32] . Treated patients who still presented three positive serologic

reactions developed fewer new electrocardiographic changes thanuntreated patients (2.1 vs 29.4%)[33] . On the other hand, a clini-cal and laboratory follow-up study of 45 Chagas patients subjectedto treatment with trypanocidal nifurtimox or benznidazole over aperiod of 10 years showed an increasing frequency of ECG altera-tions than those recorded in untreated Chagas patients, rangingfrom 67.4 to 75%; these values were not statistically different fromthose registered among treated Chagas patients (7980.5%)[34] .

Clinical trials of Chagas disease treatmentThe study of effectiveness of e tiologic treatments for Chagas dis-ease by controlled clinical assays started early in the 1960s, whena standardization of the therapeutic methodology was formulated

by the Treatment of Chagas Disease Studies Group in 1962. It was demonstrated that, in long-term therapeutic schemes, thenitrofurans are effective for the acute phase of experimentalT. cruzi infection in mice [35] . Several clinical trials were per-formed thereafter, and two compounds were proposed for cha-gasic therapy: nifurtimox and benznidazole[3638] . Currently,the treatment for chronic Chagas disease is still based on thenitrofurans, benznidazole (Rochagan and Rodanil, Roche) ornifurtimox (Lampit, Bayer), which were empirically developedthree decades ago.

Nifurtimox acts by reducing a nitro group in unstable nitro-anion radicals, producing oxygen metabolites (hydrogen anionsuperoxide and peroxide)[39] . Benznidazole acts by a differ-ent mechanism (reductive stress), which involves the covalentmodication of macromolecules by nitroreductive intermediates[39] . Nifurtimox and benznidazole also inhibit the synthesis ofDNA, RNA and proteins and enhance the degradation of thesemacromolecules by interacting with topoisomerases[40] . Bothdrugs present frequent side effects, including anorexia, vomiting,peripheral polyneuropathy and allergic dermopathy, likely due tonitroanion [40] and free oxygen radicals[39,41] . For Chagas disease,the current recommended therapy during the acute phase, recentchronic phase or congenital infection is benznidazole. Childrenand adults should be treated with benznidazole at 510 and 5 mg/kg daily, respectively, in two or three divided doses for 60 days,preferably after meals[42] . The outcome of treatment with ben-znidazole is extremely dependent on beginning the therapy assoon as the diagnostic is performed and it has been shown to beeffective in the acute phase.

Patients were grouped from clinical trials described in the liter-ature according to the disease phase : acute phase (patients treated

with benznidazole or nifurtimox 0 4 months after infection,062 years); recent chronic phase (children 014 years of age,treated with benznidazole or nifurtimox after 4 months of infec-tion); and late chronic phase (great majority of patients between15 and 69 years of age). Data from the literature described areduction in mean cure rates obtained from clinical trials accord-ing to the disease phase during treatment: congenital infection

(97.9%, treatment performed between 06 months)[4346] ; acutephase (71.5%) [4750] ; recent chronic phase (57.6%)[5159] ; andlate chronic phase (5.9%)(F IGURE 2 & T ABLE 2) .

Congenital Chagas disease is usually asymptomatic or causes

nonspecic symptoms; however, a small proportion of patientsdemonstrate severe morbidity, including low birth weight, hepato-splenomegaly, anemia, meningoencephalitis, and/or respiratoryinsufciency and high mortality[44,60] . Treatment of congenitalChagas disease induced a cure index of 93.8100% in childrenaged 06 months[4346] , 66.790.09% in chi ldren 724 months[45,61] and 12.5% in children 315 years of age(F IGURE 2 & T ABLE 2) [45] .These results show the effectiveness of congenital Chagas diseasetreatment and support the need to screen all pregnant womenand children in endemic areas to decrease or eliminate the riskof disease progression and later complications[62] .

Clinical trials performed in patients that acquired postnatalChagas disease were grouped in three cases: patients treated dur-

ing the acute phase; patients treated during the recent chronicphase; and patients treated during the late chronic phase. Duringthe acute phase of Chagas disease, 66.776.2% of parasitologicalcures were observed(F IGURE 2 & T ABLE 2) [4750] .

Clinical trials using benznidazole in children (014 years old) with recent chronic Chagas disease have shown cure rates between8.3 and 93.9% [52,5459] . However, in one study, only 8.35% (oneout of 12) of children 712 years of age treated with benznidazoleduring the indeterminate form of Chagas disease were cured[57] .Cured patients treated during the chronic phase can display positivetiters of antibodies as a consequence of an immunologic memory;only one out of 12 of these patients had positive PCR after treatmentand some individuals showed uncertain results from conventionalserology (ELISA, immunouorescence [IF] and hemagglutination[IH]). Clinical trials performed during the late chronic phase ofChagas disease with nifurtimox or benznidazole displayed low orno activity; cure rates were 019.1%(F IGURE 2) [3234,6367] . Owing tothe risk of cardiac insufciency and death during the chronic phaseof Chagas disease, specic treatment (benznidazole and nifurti-mox) can be the key to blocking the progression or appearance ofmyocardium lesions. However, the efcacy of trypanocidal treat-ment in preventing the development of chronic Chagas disease iscontroversial. Treatment of patients with mild or moderate heartChagas disease is currently under scrutiny by the BenznidazoleEvaluation for Interrupting Trypanosomiasis (BENEFIT) trial,

which compares benznidazole to placebo in 3000 patients (aged1875 years) from several centers in Latin America[68] .

In addition, the effect of treatment on the parasite load duringchronic infection is controversial and difcult to demonstrateowing to the limited serological and parasitological methodsavailable for cure detection. Cure assessment in clinical trialsof Chagas disease is evaluated through parasitological (hemo-culture, xenodiagnosis and PCR) and serological methods.

Antibodies can be detected by conventional serology (CS; ELISA,IH, IF and complement xation) and lysis-mediated comple-ment/LMCo, which detected lytic antibodies (mediates comple-ment lysis ofT. cruzi binding to a 160-kDa surface glycoprotein)[6973] . Long-term follow-up studies of some treated patients have

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demonstrated that CV assays may remain positive for severalyears despite repeated negative hemoculture, PCR and xenodi-agnosis [73,74] . Treated patients that showed negative results ofparasitological and LMCo and positive titers of CS are termeddissociated patients; experimental studies also supported thatthese patients were cured[75] . There is a need to nd better toolsto determine cure assessment.

Reversibility of myocarditis & cardiac brosis in anexperimental modelMice infected with the 21SFT. cruzistrain and treated with ben-znidazole during the chronic phase showed a reduction in inam-matory markers and in brotic alterations of the interstitial matrix,suggesting that parasite clearance may be benecial to reducestructural derangements. Conversely, animals infected with the

Figure 2. Summary of clinical trials results, according to the phase of Chagas disease when the treatment was carried out. (AC) Congenital transmission, acute phase (patients treated with benznidazole [Bz] or nifurtimox [Nfx] 04 months after infection,062 years of age); recent chronic phase (children 014 years of age, treated with Bz or Nfx after 4 months of infection); and latechronic phase (great majority of patients between 15 and 69 years of age). (A) Mean cure rate obtained in the clinical trials performed.

(B) Treatment of congenital infection; children were grouped according to age: 06 months, 7 months2 years and 315 years of age.

(C) Results from clinical trials performed during the acute, recent chronic and late chronic phase.The cure rate was determined by: dissociated patient (negative lysis mediated by complement [LMCo], parasitologic tests [PT] andpositive conventional serology [CS]) plus patient with negative result of LMCo, PT and CS.

97.9

78.4

12.5

71.5

58.6

5.9

0

100

80

60

40

20

Congenital Postnatal

M e a n c u r e r a

t e ( % )

3 1 5 y

e a r s

7 2 4 m

o n t h

s

0 6 m o n

t h s

C h r

o n i c

R e c e

n t c h r

o n i c

A c u t

e0

100

80

60

40

20

Congenital Chagas disease

C u r e r a

t e ( % )

06months

315years

724months

0

100

80

60

40

20

Chronicphase

Recentchronic phase

Acutephase

C u r e

r a t e ( % )

a

qp

on

ml

k

j

i

g

h

fe

cb

d

Postnatal Chagas disease aShikanai-Yasuda et al. [47]bAndrade et al. [48]cBahia Oliveira et al. [49]dCanado [50]eAndrade et al. [52]fSosa Estani et al. [55] , Sosa Estani and Segura [56]gSilveira et al. [53]hStreiger et al. [58]iEscriba et al. [59]

j

Ferreiraet al.

[63]kViotti et al. [33]lLauria-Pires et al. [34]mFabbro-Suasnabar et al. [64]nBraga et al. [65]oFerreira et al. [66]pFernandes et al. [67]qLana et al. [32]

aRussomando et al. [43]bBlanco et al. [44]cSchijman et al. [45]dBurgos et al. [46]eSchijman et al. [45]fChippaux et al. [61]gSchijman et al. [45]

a

g

f

e

cb

d



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Colombian strain and treated with benznidazole had myocardi-tis and brosis[76] , which was considered to result from parasiteantigen sequestration within dendritic cells in the heart tissue of

benznidazole-treated and uncured mice[77] . Similar results wereobserved in mice infected with the Y strain and treated during theacute phase with benznidazole and pozaconazole; the increase in

Table 2. Results from Chagas disease-specic clinical trials performed with benznidazole and nifurtimox,according to prenatal infection (congenital disease) and postnatal infection (acute, recent chronic andchronic phase).

Cure criteria (following timeof infection) Patient age Drug, therapeutic schedule Total patients/cured, n (%) Ref.

Congenital disease

ELISA, IF, PCR, Hc (4 years) 022 months Bz: 7 mg/kg/day, 60 days 6/6 (100%) [43]

IH, ELISA, MIC (2 years) 012 months Bz: 10 mg/kg/day, 60 daysNfx: 5 mg/kg/day, 30 days

30/32 (93.8%) [44]

IH, ELISA, PCR (23 years) 015 years Bz: 1015 mg/kg/day, 60 daysNfx: 58 mg/kg/day, 60 days

06 month olds: 10/10 (100%)7 month2 year olds: 4/6 (66%)315-year olds: 3/15 (12.5%)

[45]

MIC, ELISA, IF, PCR (7 months) 030 days Bz: 5 mg/kg/day, 60 days 2/2 (100%) [46]

CSP, ELISA (12 months) 1 day Bz: 2.5 mg/kg/day, 60 daysBz: 7.5 mg/kg/day, 30 days

101/111 (91%) [61]

Acute phaseXeno, IF, IH, IF, CMLo (2 months11 years) 062 years Bz: 410 mg/kg/day, 3060 days 12/16 (75%) [47]

CF, IF, Xeno (6 months) 640 years Bz: 510 mg/kg/day, 60 days 6/9 (66.7%) [48]

CMLo, ELISA, IH, IF, Hc, PCR (1430 years) 1875 years Bz and Nfx/dosage and daysnot informed

17/25 (68%) [49]

CF, IF, IH, ELISA (1321 years) 060 years Bz: 510 mg/kg/day, 32 days 16/21 (76.2%) [50]

Recent chronic phase

CL-ELISA (3 years) 712 years Bz: 7.5 mg/kg/day, 60 days 47/53 (64.7%) [51,52]

F29-ELISA (7 years) 612 years Bz: 5 mg/kg/day, 60 days 38/55 (69%) [55,56]

ELISA, IF, IH Xeno, PCR (820 years) 712 years Bz: 57 mg/kg/day, 60 daysNfx: 7 mg/kg/day 60/90 days

1/12 (8.3%) [53]

IF, IH, DA, Chagas test (424 years) 114 years Bz: 5 mg/kg/day, 30 daysNfx: 1215 mg/kg/day, 4560 days

29/49 (59%) [58]

ELISA, Chagas test (3 years) 013 year s Bz: 7.5 mg/kg /day, 60 days 215 /229 (93.9%) [59]

Late chronic phase

IF, ELISA, xeno (717 years) ND Bz: 58 mg/kg/day, 60 daysNfx: 1015 mg/kg/day, 60120 days

5/45 (10%)3/47 (6%)

[63]

IF, IH, CF (513 years) 2059 years Bz: 5 mg/kg/day, 30 days 19/70 (19.1%) [33]

PCR (10 years) 3160 years Bz or NFx: 10 mg/kg/day, 2060 days 0/91 (0%) [34]

IF, IH, xeno, DA (823 years) 1352 years Bz: 5 mg /kg /day, 30 daysNfx: 58 mg/kg/day, 60 days

3/63 (4.8%) [64]

PCR (ND) ND Bz or NFx: 10 mg/kg/day, 3060 days 0/17 (0%) [65]

CF, IF, IH, ELISA (618 years) 969 years Bz: 510 mg/kg/day, 40 days 9/113 (8%) [50]

PCR, Hc, IF, Chagas test (3 years) 1742 years Bz: 5 mg/kg/day, 60 days 4/80 (5%) [67]

ELISA, IH, PCR (ND) 637 years Bz: 510 mg/kg/day, 4060 days 0/28 (0%) [32] Dissociated patients were considered cured.Bz: Benznidazole; CF: Complement xation; Chagas test: Recombinant ELISA; CL-ELISA: Chemiluminescent ELISA with a trypomastigote mucin antigen;CMLo: Complement-mediated lysis; CSP: Chagas Stat-Pak; DA: Direct agglutination; F29-ELISA: ELISA using Trypanosoma cruzi agellar calcium-binding protein(F29) as antigen; Hc: Hemoculture; IF: Immunouorescence; IH: Indirect hemagglutination; MIC: Microhematocrit; ND: Not described; Nfx: Nifurtimox;Xeno: Xenodiagnostic.



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bronectin deposition was completely prevented, resulting in anexpression pattern similar to that observed in uninfected mice[78] .Our group also demonstrated that mice infected with the Y strain,treated with benznidazole and an nitric oxide (NO) donor and

considered cured showed an absence of myocarditis and a normal-ization of cytokine production, while uncured animals displayeda reduction of myocarditis compared with infected and untreatedanimals [79] . Mice infected with the Colombian strain and treated

with benznidazole during the chronic phase of infection had signi-cantly lower cardiac conduction disturbances than untreated mice,including intraventricular conduction disturbances, atrioventricularblocks and extra systoles[80] .

Mice treated with benznidazole during the acute phase of infec-tion with Y, ABC, VL-10, Be-78 and AAS strains ofT. cruzidemonstrated the benet of early treatment by displaying immedi-ate and permanent suppression of parasitemia and mortality ratereduction. The specic treatment was not efcient to reduce the

degree of heart inammation in animals inoculated with AASand VL10-resistantT. cruzistocks. However, it was observed thatthe brotic area was similar to or smaller than that observed inuntreated animals infected with AAS and VL-10 strains. Miceinoculated with strains ofT. cruzi that are susceptible to benz-nidazole (Y, Be-78 and ABC), as well as benznidazole-treatedand cured mice displayed a pattern of heart cellularity and col-lagen deposition similar to uninfected mice[81] . Similar results

were described for mice treated with benznidazole and TAK-187(Takeda Chemical Industries, Japan; ergosterol biosynthesisinhibitors) during the acute phase of infection with the Tulahuenstrain, which showed reduced myocarditis and cardiac brosis[82] .

Benet of Chagas disease therapy during the chronicphase is uncertainCurrent clinical evidence regarding whether etiologic treatmentis benecial in avoiding, stopping or reverting heart pathology inindeterminate and cardiac chronic patients is contradictory andis insufcient to support the routine use of etiologic treatment

with benznidazole. However, treatment is recommended in acute,recent chronic phases and congenital infections. Some clinicaltrials have failed to demonstrate that the treatment with benz-nidazole is able to induce a parasitological cure; thus, treatmentdoes not provide a benet to the patient. However, other data hasshown that Chagas patients treated with benznidazole during thechronic phase had less clinical and electrocardiographic progresstowards cardiomyopathy[64,78,8385] .

It has been shown that chronic Chagas patients treated withbenznidazole and nifurtimox had parasite persistence and pro-gressive electrocardiographic alterations, similar to untreated con-trol patients [34,65] . However, these clinical trials were conducted

with a treatment time of 20 and 30 days only and it is not clear ifthis short time of treatment has destructive effects on the parasite.Several authors have shown, and the Chagas disease consensus hasrecommended, benznidazole treatment for 60 consecutive days at510 mg/kg/day. However, children treated with this therapeuticschedule during the recent chronic phase of infection showed nodifference in cardiac alterations between treated and untreated

groups. ECG examinations showed a left anterior hemiblock orright bundle branch block level II in 5.0% of the placebo andbenznidazole-treated group. After 48 months, changes in ECGs

were detected in 2.5% of the placebo and benznidazole-treated

group, indicating that there are no differences between the treatedand untreated groups [55] . Similar results were described in chil-dren treated during the recent chronic phase of Chagas disease,showing a progressive chronic cardiomyopathy with crescent ECGalterations in the placebo- and benznidazole-treated groups[57] .

A total of 22 indeterminate patients and six with the cardiacor digestive forms of Chagas disease were treated with benzni-dazole; it was observed that 82.1% of treated patients (23 out of28) remained clinically stable and 95.4% of the indeterminate(21 out of 22) and 33.3% of the cardiac and digestive (two outof six) patients showed unaltered physical and laboratorial exami-nations [32] . Other studies showed that Chagas patients treated

with benznidazole during the chronic phase showed less clinical

and ECG progress towards cardiomyopathy[33,64]

. School chil-dren treated during the recent chronic phase of infection with57 mg/kg/day for 60 days displayed ECG changes similar tountreated patients. ECG recordings at the end of the follow-up(3 years after treatment) showed one (1.7%) incident of completeright bundle branch block in the benznidazole group and four(6.9%) in the untreated patients [52] .

Patients treated with benznidazole for 30 days who displayedthree positive serologic reactions (follow-up 513 years) wereconsidered not cured and developed fewer new ECG changesthan untreated patients (2.1 vs 29.4%)[33] . Moreover, in anotherclinical trial performed by these authors, patients with three posi-tive results in serologic tests forT. cruziand no evidence of heartfailure received benznidazole on the same therapeutic schedule.

After a median follow-up of 9.8 years, 14.1% of untreated patientsand 4.2% of treated patients had progression of heart disease[86] .These encouraging results suggest that chronic chagasic patientsbeneted from trypanocidal treatment, at least in terms of parasiteburden reduction [87] . However, treatment of 91 chagasic patients

with benznidazole at 10 mg/kg/day during 2060 days, followedby 10 years of clinical and laboratory observations, showed that nobenet was achieved; progressive ECG alterations were recordedin treated and untreated Chagas patients[34] .

There is a gap in knowledge regarding the benet of Chagasdisease therapy during the chronic phase of infection in patientsdisplaying the indeterminate, cardiac and digestive forms of thedisease. The BENEFIT trial is an international, multicenter, dou-ble-blind, placebo-controlled trial of benznidazole trypanocidaltreatment in patients with chronic Chagas heart disease that aimsto test the hypothesis that trypanocidal therapy is benecial forpatients with chronic Chagas heart disease[87] .

Inhibitors of ergosterol biosynthesisExperimental and clinical data supported the idea that etiologictreatment reduces or eliminates cardiac damage during the acuteand chronic phases of infection; thus, the discovery of new effec-tive compounds could be used in the clinical trial of indeterminateand cardiac chronic patients to avoid, reduce or stabilize cardiac



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damage. Moreover, it would be important for patients infected withbenznidazole-resistant strains and for those intolerant to this drug.Over the years, several varieties of drugs have been investigated todevelop safe and efcient chemotherapy for Chagas disease. These

include natural products, cruzain (primary cysteine protease),ruthenium nitric oxide donor compounds (deliver nitric oxideto T. cruzi -infected cells), inhibitors and substrates of the purinesalvage pathway, cysteine protease inhibitors, pyrophosphatasemetabolism inhibitors and sterol biosynthesis inhibitors, amongother rational approaches[79,8890] . Four classes of sterol biosynthe-sis inhibitors were testedin vitroand/or in experimental infectionagainstT. cruzi : squalene synthase inhibitors, squalene epoxidaseinhibitor, oxidosqualene cyclase inhibitors and C14a -demethylaseinhibitors (ergosterol biosynthesis inhibitors). Squalene synthaseinhibitors, squalene epoxidase inhibitor and oxidosqualene cyclaseinhibitors disrupt the steps of ergosterol biosynthesis that are com-mon in cholesterol biosynthesis; their use in humans could be dan-

gerous due to side effects on the host hepatic tissue. However, afterlanosterol synthesis, the subsequent steps in cholesterol or ergos-terol biosyntheses are distinct, involving the production of dis-tinct compounds and the participation of specic enzymes. Azolescomprise imidazole and triazole derivatives that act at the levelof cytochrome P450-dependent C14a sterol demethylase[89,90] .Recent data from high-throughput screening studies have shownseveral new compounds with biological activity againstT. cruzi .These include nonpeptidic cruzain inhibitors, blockers of the sterol14a -demethylase enzyme, arylimidamide compounds related topentamidine, derivatives of nifurtimox and ruthenium complexes,among others[90] . The biological activity of azoles againstT. cruzi is thought to be related to their ability to inhibit the biosynthesis ofsterols through inhibition of cytochrome P450 sterol demethylase. T. cruzihas an absolute necessity for specic endogenous sterolsfor cellular viability and proliferation and is extremely sensitive toergosterol biosynthesis inhibitorsin vitro[41,9193] . Moreover, thesecompounds have special pharmacokinetic properties in mammals,including large volumes of distribution, long half-lives and lowertoxicity[94,95] .Miconazole and econazole inhibitedT. cruzigrown in vitro and modied its ergosterol constitution[41] . Itraconazoleand ketoconazole induced a parasitological cure in infected mice

with a small number ofT. cruzi strains [9699] . Ketoconazole iseffective againstT. cruzi in vitroand during acute experimentaltreatment of mice. However, itraconazole and ketoconazole are notefcient during chronic experimental infection[100] or in chronicChagas patients[101] . ICI-195739 and D0870 are other tested azolederivatives. Treatment with ICI-195739 (1 mg/kg/day for 15 days)of acutely infected mice withT. cruzicontrolled parasitemia andmortality and generated a 50% cure rate, which could probablybe enhanced with a longer treatment period[102] . D0870 gener-ated a 75100% and 6070% parasitological cure rate during theacute and chronic phases, respectively. D0870 development wasabandoned owing to variation of drug half-life in mice and lowtolerance of animals to this drug[103] .

Recently, it was shown that ravuconazole, TAK-187, albacon-azole and pozaconazole are capable of inducing high cure ratesin experimentally infected mice during the acute and chronic

phases and in dogs infected during the acute phase of infec-tion with a T. cruzistrain (Y) partially resistant to benznidazoletherapy [8385,104106] .

The compound named TAK-187 showed a MIC of 1 nM over

intracellular forms ofT. cruzi. Acute treatment of infected mice with Cl (benznidazole susceptible), Y (benznidazole partia llyresistant), Colombian, SC-28 and VL-10 (benznidazole resistant)strains induced 100, 78100, 6075, 100 and 50% cure rates,respectively, and no side effects were observed at 20 mg/kg/day[84,105] . Albaconazole (Uriach & Company) is an experimentalazole with potent and broad-spectrum antifungal activity anda remarkably long half-life in dogs (51 h), monkeys (24 h) andhumans (120 h); however, the half-life is short in mice (0.5 h)[107109] . The MIC to amastigote forms ofT. cruziis 10 nm [110] . Wetested the activity of a lbaconazolein vivo using a canine modeltreated during the acute infection phase with 1.5 mg/kg body-

weight per day for 60, 90 and 150 days. The animals infected

with the Y strain showed 25 and 100% cure rates when treatment was administered for 60 and 90 days, respectively. Berenice-78-infected animals did not display parasitological cure, but thetreatment suppressed 100% of mortality and parasitemia[85] .

Although natura l resistance to albaconazole was detected, thecompound is still a possible therapeutic approach to Chagas dis-ease in humans owing its long half-life (120 h). The half-life ofalbaconazole appears to be longer in humans than in experimen-tal animals, therefore additional clinical tests are required inorder to accurately asses its pharmacokinetic properties.

Ravuconazole displayed a MIC of 1 nm to amastigotes (intra-cellular forms of the parasite) and a long terminal half-life inhumans (48 days). The treatment of acutely infected mice withthe Y or CL strain (partially resistant and susceptible to benzni-dazole) had 100% curative activity; however, the animals infected

with the Colombian strain were resistant to ravuconazole treat-ment, consistent with its short terminal half-life in mice (4 h)[111] .Ravuconazole administered during the acute phase of experimen-tal infection in dogs with Y and Berenice-78T. cruzistrains was

well tolerated, as no signicant side effects were observed duringtreatment with 6 mg/kg twice a day (12 mg/kg/day) for up to90 days. Suppression of parasitemia was observed in all treatedanimals in the rst day, but no curative activity was observedin acute Chagas disease. In spite of its intrinsic potent activityagainst T. cruzi , the lack of curative effects is possibly due to ashort (8.8 h) terminal half-life in dogs. In humans, however, thehalf-life is signicantly longer (48 days)[E C . L , D ] for stimulated chemotherapy with ravuconazole in Chagas diseasepatients. The Drugs for Neglected Diseases Initiative (Geneva,Switzerland) announced an agreement with Eisai Co. Ltd for theclinical development of E1224, a water-soluble prodrug (mono-lysine derivative) of ravuconazole for clinical trials on chronicChagas disease, which was estimated to begin in 2010[106,112] .

Pozaconazole has been shown to have high trypanocidal activ-ity in vitro and it induced a cure in mice during the chronic andacute experimental infections. Cure rates were 100 and 50% foranimals administered with acute (Y strain: 25 mg/kg/day) andchronic strains (Bertoldo strain: 15 mg/kg/day) for 28 days,



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Key issues

Chagas disease affects 7.7 million people and 28 million are at risk of acquiring it in Latin America. The cardiac form of the disease is characterized by dilated cardiomyopathy, which starts with an acute myocarditis, followed by

myocardial remodeling, brosis, arrhythmias, cardiac dilatation and heart failure. The mechanisms of pathogenesis during chronic Chagas disease are diverse and appear to involve the concerted participation of

parasite persistence, deregulated immune responses, as well as microcirculatory and autonomic disturbances. Currently, the treatment for chronic Chagas disease is still based on benznidazole or nifurtimox, both of which induce severe side

effects and have low cure rates (~5.9%). Clinical trials have failed to demonstrate the role of current treatments in slowing or preventing the development of disease progression

in chronically infected individuals. The majority of experimental data indicated that early treatment is benecial to avoid future cardiac damage. The potential clinical advantage of the treatment with benznidazole in patients with mild or moderate Chagas heart disease is currently

under scrutiny by the Benznidazole Evaluation for Interrupting Trypanosomiasis (BENEFIT) trial in several centers in Latin America. Azole derivatives are a promising compound class for chronic Chagas disease therapy.

respectively[83] . No signicant cure rates were observed in thesame experiment when treated with benznidazole. Pozaconazoleremains a good candidate for clinical trials of chronic Chagas dis-ease. Acutely infected mice with Cl, Y and Colombian strains and

treated with pozaconazole displayed 100, 90 and 50% parasitologi-cal cure rates, respectively. Chronic treatment generated 60, 50 and50% cure rates in mice infected with Cl, Y and Colombian strains,respectively. Moreover, no parasitological cure was observed inmice infected with theseT. cruzistrains and treated with benznida-zole during the chronic phase[105] . Pozaconazole was efcient in aclinical case study in Spain of chronic Chagas disease and systemiclupus erythematosus treatment. While benznidazole induced areduction, it did not eliminateT. cruzi [113] . Pozaconazole was moreeffective than benznidazole for immunocompetent and immuno-suppressed hosts in controlling parasitism during acute and chronicChagas disease[78,105] . Moreover, pozaconazole reduced nonspe-cic splenocyte proliferation in the early acute phase but allowed

higher CD4 and CD8 T-cell activation in the late acute phase, when the adaptive immune response takes control of the infection.Pozaconazole has already been registered as a systemic antifungalin many countries owing to its potent and extended antifungalspectrum and its excellent safety prole in humans[90] . Theseresults support the notion that pozaconazole could be superior tobenznidazole for the treatment ofT. cruzi infection in humans[78] . It is reported that a Phase II clinical study with pozaconazolefor the treatment of Chagas disease, sponsored by Merck & Co.,is planned in Spain[90] . All azole derivatives described (TAK-187,ravuconazole, albaconazole and pozaconazole) efciently reducedcardiac damage in treated animals when compared with untreatedanimals[78,84,85,105,111] . Currently, a Phase II clinical trial is beingperformed to test the anti-infective properties of pozaconazoleand ravuconazole on Chagas disease. If successful, this will be therst study in several years to identify a novel compound for thetreatment of Chagas disease[90] .

Expert commentary We believe that the development of clinical tr ials on the assess-ment of novel strategies for the pharmacotherapy of chronicallyinfected patients is currently a necessity in the research of Chagas

disease. The majority of data available from experimental mod-els have indicated that conventional trypanocidal treatment isbenecial for late stages of the disease; however, the results fromclinical trials with chronic Chagas disease patients are dissimi-

lar, thus the efcacy of trypanocidal treatment in preventingthe development of chronic cardiomyopathy remains contro-versial. The development of novel pharmacological compoundsfor Chagas disease treatment should take into account the factthat the parasite is a fundamental element in the pathogenesisand that its elimination may prevent heart problems and deathin chronic disease; in this regard, we think that azole derivativesare among the promising compounds.

Five-year viewGiven the cumulative data, it is possible to consider that, in thenext 5 years, the research in the chemotherapy of Chagas disease

will provide novel compounds derived from the current intensive

experimental research on targets for both the parasite as well asimmune modulators.In addition, it is expected that the BENEFIT trial will pro-

vide denitive information about the clinical advantage of treat-ing chronic patients with benznidazole. New clinical trials withchronic patients may also be conducted using azole derivatives,such as albaconazole and pozaconazole.

Unfortunately, however, it is possible that the lack of interest frompharmaceutical industries will persist, making it difcult to translatethe experimental data into novel clinically available compounds.

Financial & competing interests disclosureThis work was supported by Fundao de Amparo Pesquisa do Estadode So Paulo (FAPESP), Conselho Nacional de DesenvolvimentoCientfico e Tecnolgico (CNPq) and Coordenao de Aperfeioamentode Pessoal de Nvel Superior (CAPES). The authors have no otherrelevant aff iliations or financial involvement with any organization orentity with a financial interest in or f inancial conflict with the subjectmatter or materials discussed in the manuscript apart from thosedisclosed.

No writing assistance was utilized in the production of thismanuscript.



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Website

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