pathogens Article Benznidazole Treatment: Time- and Dose-Dependence Varies with the Trypanosoma cruzi Strain Kátia da Silva Fonseca 1,† , Luísa Perin 1,† , Nívia Carolina Nogueira de Paiva 1 , Beatriz Cristiane da Silva 1 , Thays Helena Chaves Duarte 2 , Flávia de Souza Marques 2 , Guilherme de Paula Costa 1 , Israel Molina 1,3 , Rodrigo Correa-Oliveira 1,4 , Paula Melo de Abreu Vieira 1,2 and Cláudia Martins Carneiro 1,5, * 1 Laboratory of Immunopathology, Nucleus of Biological Sciences Research, Federal University of Ouro Preto, Ouro Preto 35400-000, Brazil; katia.fonseca@gmail.com (K.d.S.F.); perindemelo@gmail.com (L.P.); niviacarolinanp@ufop.edu.br (N.C.N.d.P.); luisaperin@yahoo.com (B.C.d.S.); guilherme.costa@aluno.ufop.edu.br (G.d.P.C.); israelmolina@ymail.com (I.M.); correa@cpqrr.fiocruz.br (R.C.-O.); paula@ufop.edu.br (P.M.d.A.V.) 2 Laboratory of Morphopathology, Department of Biological Sciences, Nucleus of Biological Sciences Research, Institute of Exact and Biological Sciences, Federal University of Ouro Preto, Ouro Preto 35400-000, Brazil; thays.duarte@aluno.ufop.edu.br (T.H.C.D.); flavia.marques1@aluno.ufop.edu.br (F.d.S.M.) 3 Tropical Medicine and International Health Unit, Department of Infectious Diseases, Vall d’Hebron University Hospital, Universitat Autònoma de Barcelona, PROSICS Barcelona, 08035 Barcelona, Spain 4 Laboratory of Cellular and Molecular Immunology, René Rachou Research Center, Oswaldo Cruz Foundation, Belo Horizonte 30190-002, Brazil  5 Department of Clinical Analysis, School of Pharmacy, Federal University of Ouro Preto,  Ouro Preto 35400-000, Brazil Citation: Fonseca, K.d.S.; Perin, L.; * Correspondence: carneirocm@ufop.edu.br; Tel.: +55-31-3559-16-94; Fax: +55-31-3559-16-80 de Paiva, N.C.N.; da Silva, B.C.; † Kátia da Silva Fonseca and Luísa Perin contributed equally to this work. Duarte, T.H.C.; Marques, F.d.S.; Costa, G.d.P.; Molina, I.; Correa-Oliveira, R.; Abstract: As the development of new drugs for Chagas disease is not a priority due to its neglected Vieira, P.M.d.A.; et al. Benznidazole disease status, an option for increasing treatment adherence is to explore alternative treatment Treatment: Time- and Dose- regimens, which may decrease the incidence of side effects. Therefore, we evaluated the efficacy Dependence Varies with the of different therapeutic schemes with benznidazole (BNZ) on the acute and chronic phases of the Trypanosoma cruzi Strain. Pathogens disease, using mice infected with strains that have different BNZ susceptibilities. Our results show 2021, 10, 729. https://doi.org/ that the groups of animals infected by VL-10 strain, when treated in the chronic phase with a lower 10.3390/pathogens10060729 dose of BNZ for a longer period of time (40 mg/kg/day for 40 days) presented better treatment efficacy than with the standard protocol (100 mg/kg/day for 20 days) although the best result in the Academic Editor: Vyacheslav Yurchenko treatment of the animals infected by the VL-10 strain was with100 mg/kg/day for 40 days. In the acute infection by the Y and VL-10 strains of T. cruzi, the treatment with a standard dose, but with a Received: 10 April 2021 longer time of treatment (100 mg/kg/day for 40 days) presented the best results. Given these data, Accepted: 13 May 2021 our results indicate that for BNZ, the theory of dose and time proportionality does not apply to the Published: 9 June 2021 phases of infection. Publisher’s Note: MDPI stays neutral Keywords: chagas disease; Trypanosoma cruzi; benznidazole; therapeutic strategies; mice with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Chagas disease is a neglected protozoan disease that affects more than 6 million people worldwide and is endemic in Latin America [1]. It is caused by the hemoflagellate Copyright: © 2021 by the authors. parasite Trypanosoma cruzi (T. cruzi), belonging to the order Kinetoplastida and family Licensee MDPI, Basel, Switzerland. Tripanosomatidae, whose main invertebrate hosts and vectors of Chagas disease, are This article is an open access article hematophagous insects of the subfamily Triatominae [2,3]. T. cruzi has a high genetic distributed under the terms and heterogeneity and using different molecular markers, genotyping of the strains of the conditions of the Creative Commons parasite was carried out, in order to classify it in several subgroups (or discrete typing Attribution (CC BY) license (https:// units, DTUs) termed TcI–TcVI. However, studies have suggested that the DTUs would creativecommons.org/licenses/by/ have the status of subspecies [3]. 4.0/). Pathogens 2021, 10, 729. https://doi.org/10.3390/pathogens10060729 https://www.mdpi.com/journal/pathogens Pathogens 2021, 10, 729 2 of 10 Benznidazole (BNZ), the reference drug in the treatment of Chagas disease, shows satisfactory results in the acute and recent chronic phases of infection, but causes numerous side effects that lead to treatment interruption by significant numbers of patients [4–10]. There are few efforts to discover new drugs for Chagas disease, therefore, an option for increasing treatment adherence is to explore alternative treatment regimens with BNZ, which could decrease the incidence of side effects. A number of studies have suggested that it is possible to reduce the dose of BNZ currently used for treatment of Chagas disease. First, researchers reported that 20% of adults who prematurely abandoned BNZ treatment due to adverse effects, displayed negative serology for T. cruzi [11]. Furthermore, although plasma concentrations of BNZ in children subjected to the treatment are lower than in adults, the treatment efficacy is proven, suggesting that the BNZ dose used in the treatment of adults could be decreased and still generate similar exposure to that observed in children, maintaining efficacy and reducing side effects [12]. A prospective study of population pharmacokinetics showed, through dose simulations, that it is possible to decrease the dose of BNZ in relation to the conventional dose while keeping the plasma concentration within the recommended target range, indicating the need to evaluate whether lower doses could be as efficient as the conventional dose [13]. Moreover, animals subjected to intermittent treatment that reduces the total dose of BNZ can be cured of T. cruzi infection [14]. However, a recent study showed that to obtain parasitological cure in mice chronically infected with resistant T. cruzi strains it is necessary to increase the dose of BNZ and extend the length of treatment [15]. Lastly, mice in the acute phase of T. cruzi infection, when treated with BNZ, have a dependent dose-time response profile [16]. It still remains necessary to evaluate other therapeutic regimens by altering dose and time of treatment simultaneously and, mainly, to perform the evaluation in the chronic period of the infection, the phase when most patients are treated. Therefore, the objective of this work was to evaluate the influence of the dose and time treatment in curing mice in the acute and chronic phases of the infection against strains with different susceptibilities to BNZ treatment. 2. Materials and Methods 2.1. Animals and Ethics One hundred and twenty Swiss mice, 30 days old, weighing 18–25 g, and provided by the Animal Science Center of the Federal University of Ouro Preto (CCA, UFOP), were kept under control conditions with regular cycles of light, temperature of 23 ± 2 ◦ C, and availability of food and water ad libitum. All procedures were performed according to ethical principles recommended by the National Council for Control of Animal Experimen- tation (CONCEA) and approved by the ethics committee of Federal University of Ouro Preto (Protocol CEUA-UFOP number: 2016/33). The mice were monitored daily by the researchers, who have extensive experience, so that visible physical changes were communicated to the veterinarian responsible for the sector, in order to determine the appropriate handling of the animal. We emphasize that some physical changes are characteristic of infection by Trypanosoma cruzi, such as weight loss and bristly fur, and in none of the evaluations carried out by the veterinarian throughout the project, were the animals euthanized. 2.2. Therapeutic Schemes Mice were inoculated with 5 × 103 of the blood trypomastigote forms of T. cruzi, all trypomastigotes were obtained from the blood samples of infected albino Swiss mice at the peak of parasitemia. These animals were divided into three groups: infected with Y strain (DTU II) and treated in the acute phase (n = 40); infected with VL-10 strain (DTU II) and treated in the acute phase (n = 40); and infected with VL-10 strain and treated in the chronic phase (n = 40). To exclude variables, the inoculum was the same for all phases/strains. Y Pathogens 2021, 10, 729 3 of 10 strain was not observed in chronic phase because the animals are not able to survive until chronic phase with this inoculum. In the acute phase and in the chronic phase of this experiment, groups of eight animals were analyzed: (1) infected and untreated; (2) infected and treated with BNZ 100 mg/kg/day for 20 days; (3) infected and treated with BNZ 100 mg/kg/day for 40 days; (4) infected and treated with BNZ 40 mg/kg/day for 20 days; and (5) infected and treated with BNZ 40 mg/kg/day for 40 days. BNZ was prepared by pulverization of 100 mg tablets (Pharmaceutical Laboratory of Pernambuco State, Recife, Brazil), suspended in an aqueous solution of methylcellulose 0.5% (Sigma-Aldrich, St. Louis, MO, USA), and each animal received, according to body weight, the suspension of the drug by gavage. The treatment was initiated in the acute phase after confirmation of infection. The mice in the untreated group received oral gavage with water and the treated groups received BNZ suspended in a solution of water and methylcellulose. As this drug has low solubility in water [17], this strategy is necessary to enable greater homogeneity of the suspended substance, being the methylcellulose the suspending agent most used in pharmaceutical preparations, including in studies carried out by Chagas disease research groups [16,18,19]. Animals were evaluated in relation to survival, fresh blood examination (FBE), para- sitemia, real-time polymerase chain reaction (qPCR) of blood five days after immunosup- pression with cyclophosphamide (CyI), necropsy and qPCR of heart and colon 30 days after CyI. Treatment by gavage of animals in the chronic phase was initiated 120 days after infection. The same parameters evaluated in the acute phase were used in the chronic phase. The experiments lasted from 78 days (acute phase—20 days) to 213 days (chronic phase—40 days). 2.3. Cure Criteria For therapeutic evaluation, the analysis included: survival, parasitemia, and cure through FBE and qPCR after CyI. Only animals that were negative in both tests were considered cured [20]. 2.3.1. Survival Animals were followed daily after inoculum up to 30 days after the end of the treat- ment and mortality was recorded [20]. 2.3.2. Parasitemia Parasitemia was evaluated daily from the 4th day after infection until examination was negative for five consecutive days, following the adapted methodology [21]. 2.3.3. Immunosuppression and Fresh Blood Examination Thirty days after the end of treatment all mice were subjected to CyI. Parasitemia was evaluated daily during this process and until five days after CyI, adapted methodology [22]. 2.3.4. Real-Time Polymerase Chain Reaction (qPCR) qPCR in Total Blood Peripheral blood of mice was collected five days after the end of CyI, stored in Eppendorf tubes containing EDTA (anticoagulant), and kept in ice until extraction. Samples were subjected to procedures of DNA extraction using DNeasy® Blood and Tissue Kit (Qiagen® , Hilden, Germany), following the manufacturer’s recommendations. qPCR in Tissue Fragments of heart and colon were collected during necropsy and then subjected to procedures of DNA extraction using WizardTM Genomic DNA Purification Kit (Promega, Madison, WI, USA), following the manufacturer’s recommendations. qPCR in Tissue Fragments of heart and colon were collected during necropsy and then subjected to procedures of DNA extraction using WizardTM Genomic DNA Purification Kit Pathogens 2021, 10, 729 4 of 10 (Promega, Madison, WI, USA), following the manufacturer’s recommendations. 2.4. Statistical Analyses 2.4. Statistical Analyses Statistical analyses of the data were performed using GraphPad Prisma software (GraphPad Software, Statistical analysesSan of Diego, the data CA, wereUSA). The parasitemia performed was expressed using GraphPad Prisma as mean ± software standard error. (GraphPad To compare Software, the area San Diego, CA,under USA).the The curve (AUC), Kruskal-Wallis parasitemia was expressed test/ ± Dunn’s as mean multiple error. standard comparisons To compare test the was used, the area under and Kruskal-Wallis curve test/Dunn’s (AUC), Kruskal-Wallis multiple test/Dunn’s multiple comparisons comparisons test weretest wastoused, used and Kruskal-Wallis compare test/Dunn’s maximum parasitemia multiple comparisons peak. test were The used data to compare were maximum evaluated parasitemia by contingency peak.Fisher exact test, and odds ratio to table, The data compare were evaluated the percentage byamong of cure contingency table, Fisher the different exact groups. test, andinodds Differences meanratio to values compare the percentage were considered of cure significant at pamong < 0.05. the different groups. Differences in mean values were considered significant at p < 0.05. 3. Results 3. Results Mice infected with Y strain and treated with all protocols showed a reduction in the Mice infected parasitemia withpatent peak and Y strain and treated period comparedwithtoall protocols untreated showed animals a reduction (Figure 1). in the parasitemia peak and patent period compared to untreated animals (Figure 1). Figure 1. Effect of different benznidazole (BNZ) therapeutic protocols on parasitemia by Trypanosoma cruzi strains with Figure 1. Effect of different benznidazole (BNZ) therapeutic protocols on parasitemia by Trypanosoma cruzi strains with different susceptibilities to BNZ. (A) Y: partially susceptible, and (B) VL-10: resistant. Standard treatment for mice: BNZ different susceptibilities to BNZ. (A) Y: partially susceptible, and (B) VL-10: resistant. Standard treatment for mice: BNZ 100 mg/kg/day for 20 days. Parasitemia was measured by an adapted version of the Brener method. 100 mg/kg/day for 20 days. Parasitemia was measured by an adapted version of the Brener method. Animals infected with Y strain presented a survival rate of 63% when untreated, and 0% cure rate, Table 1. Treatment with 100 mg/kg/day of BNZ improved the survival rate to 100%, with a cure rate of 75% for the 20-day treatment, which increased to an 87% cure rate when the treatment duration was extended to 40 days. However, for this strain, the lower dose of BNZ (40 mg/kg/day) resulted in no cure for either 20-day or 40-day treatments. Moreover, survival rates with 40 mg/kg/day of BNZ for 40 days decreased following treatments Untreated animals wereinfected compared: 5 of 10 with BNZ 100 mg/kg/day VL-10 strain presented for 20 similar days, BNZ 40 mg/kg/day features when the following Untreated treatments 0% cure animals were rate, Table infected compared: 1. Treatment with BNZ VL-10 100 with strain mg/kg/day 100presented mg/kg/day for 20 days, of similar BNZ BNZ improved features 40 Animalsmg/kg/day when theinfected thethesurviv with Y following treatments were compared: BNZ 100 mg/kg/day for 20 days, BNZuntreated, for 20 Animals days, and infected BNZ 40 with Y mg/kg/day strain presented for 40 days a survival (0% cure), rate Table of 63% 1. By when changing 40 mg/kg/day timeand for 0% 20 following days, cure and rate,and to TableBNZ 100%, treatments 40 1. 40 with were mg/kg/day Treatment a cure 100with rate compared: for of 40 75% BNZ days for 100 (0% the cure),20-day mg/kg/day Table treatment, for1. 20 By days, 0% which cure changingBNZ rate,increased 40 the mg/kg/day Table time 1. to an Treatm87 offor the20 standard days, protocol BNZ (BNZ mg/kg/day for100 mg/kg/day 40 mg/kg/day daysfor(0% 20 days) of BNZ cure), toTable BNZimproved 1.100 the survival Bymg/kg/day changing the rate fortime 40 oftothe days, for 100%, there 20with standardisdays, an a rate andwhen protocol cure improvement BNZ rate (BNZ ofthe 4075% treatment mg/kg/day in100 formg/kg/day the therapeutic duration forefficacy 20-day 40for dayswas 20 treatment, (0%extended days) (12%). cure), to BNZ which These to Table 40to1. 100 increased results days. Byto 100%, mg/kg/dayHowever, changing indicate with an 87% thatfor the a cure40 cure for fortime thisofstra rate 7 cted with Y strain presented a survival of ratetheofstandard 63% when protocol lower untreated, dose (BNZ and of 100 mg/kg/day for 20 days) to BNZ 100 mg/kg/day for 40 BNZ (40 mg/kg/day) resulted in no cure for either 20-day or days, rate of when the there standard is thean treatment protocol improvement duration (BNZ in 100 therapeutic was mg/kg/day efficacy extended to for 40 20 (12%). days) days. These to However, BNZ results rate 100 mg/kg/day indicate forwhen this the that strain, for treatment the for 40 dura e 1. Treatment with 2021,100 mg/kg/day ofboth BNZstrains, days, there isYan improved andthe VL-10, improvement survival treatments. the rate therapeutic in Moreover, therapeutic efficacy survival efficacyis(12%). rates time- These with 40 and dose-dependent results indicate that mg/kg/day ofdoseBNZ in the for Pathogens 10, 729 both lower acute days, strains, dose phase, thereY of i.e., is and BNZ the anefficacy improvement VL-10, (40 the mg/kg/day) increases in therapeutic therapeutic efficacy resulted proportionally inefficacy isto no time- cure the(12%). and for increase These results lower dose-dependent eitherin 20-day dose or or offor indicate length in5BNZ of40 the 40-dayof 10days that dec for mg (40 re rate of 75% for the 20-day treatment,both which strains, increased Y and with to VL-10, an 87% increase the cure in therapeutic efficacy is time- and dose-dependent in the treatment duration (100% survival for 20 days of treatment and 8 acute both phase, treatments. strains, i.e., the Y and efficacyVL-10, increasesthe therapeutic proportionally efficacy to is the time- increase and in dose-dependent dose treatments. or length Moreover, of in the surviv tment duration was extended to 40 days. treatment. acute phase,Moreover, However, i.e., for the this survival efficacy strain, therates with increases 40 mg/kg/day proportionally to the ofincrease BNZ forin40dose days ordecreased length of acute treatment. with phase, 40 days i.e., the of treatment). efficacy increases proportionally to the increase with in dose increase or in length treatment of du NZ (40 mg/kg/day) resulted in no cure forincrease treatment. either in treatment 20-day or 40-day Untreated duration (100% survival for 20 days of treatment and 88% for animals infected with VL-10the strain presented similar features wh Table 1. Time- and dose-dependence treatment. of BNZ treatment on different 40 days of treatment). over, survivalTable rates1.with 40 mg/kg/day 40 with days of BNZ of increase treatment). for in treatment 40 days durationTrypanosoma decreased (100% survival cruzi strains for 20indays acute phase of treatment of Chagas and 88% for Time- and dose-dependence of BNZ following treatment ontreatments different were Trypanosomacompared:cruzi BNZ 100 mg/kg/day strains in the acute phase for 20 when of days, BNZ Chagas Untreated the 40infec mg/ disease. eatment durationTable(100% 40days 1. Time- and dose-dependence Untreated days of treatment). oftreatment animals BNZ treatment infected on different with VL-10 Trypanosoma strain cruzi presented strains in thesimilar acute phasefeaturesof Chagasanimals disease.Table 1.survival Time- andfor 20 dose-dependence of of for and BNZ20treatment days,88% and forBNZ on different40 Trypanosoma for mg/kg/day cruzi40 strains days in (0%thecure), acute phase following Table of1.Chagas By treatments changing were coth nt). disease. following treatments were compared: BNZPositive 100 mg/kg/day qPCR for 20 days, BNZ 40 mg/kg/day disease. of the standard protocol (BNZ 100 mg/kg/day for 20 days) for 20 to days,BNZ and 100 BNZ mg/kg/day 40 mg/ imals infectedTable Time- and with1. VL-10 strain for 20 similar dose-dependence presented days, of BNZ and BNZ 40 treatment features mg/kg/day on when different theimprovementfor 40Positive Trypanosomadayscruzi (0% cure),inTable strains qPCR the acute1. By changing phase of Chagas the time Survival days, Positive there is an Positive infor qPCR therapeutic efficacy Total (12%).Positive These results disease. nts were compared: BNZ 100 mg/kg/day for 20 days, of the standard BNZ Survival protocol 40strains, (BNZ mg/kg/day Positive 100 mg/kg/day ɸ 20 Positive days)qPCR to BNZ of 100 the standard mg/kg/day Total Positive for 40indicate protocol (BN both Y and VL-10, the ɸ therapeutic efficacy is time- and dose-dependent Strain £ Group days, there Survival is an improvement Positivein therapeutic efficacy ɸ (12%). These results days,Positive Total there isCure indicate an that improvemen #for NZ 40 mg/kg/day for 40 days (0% cure), Table 1. By Survival §changing the timePositive Total Positive Strain £ £ Group both strains, Y acute and phase, Positive VL-10,FBE i.e., the the efficacy therapeutic increases Positive efficacy qPCRisɸproportionally time- and to Total both the dose-dependent increase Positive strains, Y Cure in in and #dose or le the VL-10, t Strain Strain Group Group § Cure# # Cure otocol (BNZ 100 Strain mg/kg/day £ for 20 days) Group to BNZ 100 Survival mg/kg/day §treatment. FBE for 40 Cure # mprovement in therapeutic efficacy acute (12%). phase, These resultsi.e., indicate FBE the§ efficacy ¶ FBE that increases for Blood Blood proportionally to the increase in dose or length ofefficacy Heart Heart Colon Colon acute Mice Mice phase, i.e., the Untreated 5/8 (63%) 5/5 (100%) ¶ FBE Blood * Heart - Colon - Mice treatment. 5/5 (100%) 0 d VL-10, the therapeutic BNZ 100 efficacy mg/kg/day—20 treatment. is time- days and 8/8dose-dependent (100%) 0/8 (0%) in the¶ 1/8Blood ¶ (13%) 1/8 Heart (13%) Colon 2/8 (25%) Mice 2/8 (25%) 75 a,d,e Y BNZ 100 Untreated Table 1. Time- and dose-dependence mg/kg/day—40 days 5/8 (63%) 5/5 of (100%) 8/8 (100%) * Blood BNZ treatment on different Heart - Colon - Trypanosoma 1/8 (22%) cruzi strains 5/5 (100%)Mice in the acute0a,b,d,e 0/7 (0%) phase of Chag 0/8 (0%) 0/8 (0%) 1/8 (13%) 87 he efficacy increases proportionally disease.to days Untreated the increase in dose or 5/8(100%) (63%) 5/5length (100%) of 1/1 * -1. Time-2/8 -(100%) 5/5 8/8(100%) 75 0a,d,e 0 BNZ Table 1.BNZ 40mg/kg/day—20 100 Time-mg/kg/day—20 and days dose-dependence Untreated BNZ 40 mg/kg/day—40 days 8/88/8 of(100%) BNZ 5/8 7/8 (88%) 7/8 0/8 treatment (63%) (88%) 7/75/5 (0%) on 1/8(100%) different (100%) (100%) (13%) 1/7(13%) Table 1/8 Trypanosoma - * (14%) cruzi - strains 0/7 (0%) 7/7 andin dose-dependence (25%) -the acute2/8 7/7 (100%) (100%)of BNZ (25%) phase 5/5 of Chagas 7/7 (100%) (100%) treatment 0b 0a,d,e on d BNZ 100 100 mg/kg/day—20 Untreateddays 8/8 8/8 (100%) (100%) 5/8 (63%) 0/8(22%)(0%) 5/5 (100%) 1/8 (13%) * 1/8 (13%) - 2/8 (25%) - 1/8 2/8 (13%) (25%) 5/5 (100%) 75a,b,d,e 0 Y disease. BNZ BNZ 100mg/kg/day—40 mg/kg/day—20days days 8/8 (100%) 1/80/8 (0%) 0/7 1/8-(0%) (13%) disease. 0/8 1/8(0%) (13%) 0/8 2/8 (0%) (25%) qPCR Positive 2/8 (25%) 8775a,b,d,e a,d,e Y Pathogens BNZ BNZ 100 BNZ 40 mg/kg/day—40 100 mg/kg/day—20 Untreated mg/kg/day—20 days days days 8/8 8/8 8/8(100%) (100%) (100%) 8/8 (100%)8/8 1/8 7/8 (22%) (100%) (88%) 0/8 (0%) 1/10/7 (0%) (100%) 1/8 (13%) 0/8 8/8 1/7 (0%) (100%) (14%) 1/8 (13%)7/70/8 8/8(0%) 2/8 (100%) (100%) (25%) 1/8 8/8 8/8(13%) (100%) 2/8 (100%) (25%) 87 0 0 75 a,d,e Y 2021, BNZ10, x100 BNZ FOR 100 PEER REVIEW mg/kg/day—40 mg/kg/day—20 daysdays 7/8 8/8 (88%) (100%) 6/71/8 (88%) (22%) 0/1 0/7(0%) (0%) 0/8(71%) 5/7 (0%) 0/8 (0%) 7/7 (100%) 1/8 (100%) 7/7 (13%) 587 of0a,b,d,e 10 atment on different VL-10 Pathogens Trypanosoma BNZ YBNZ 2021, 10,40 BNZ 40 BNZ x100 FOR 100cruzi mg/kg/day—20 PEER mg/kg/day—40 mg/kg/day—40 strains mg/kg/day—40 REVIEW in the 8/8 days days days acute days 8/8 7/8 (100%) phase (100%) (88%) of Chagas 8/8 (100%) 7/8 7/7 5/8 (100%) (63%) Survival (88%) 1/8 (22%) 1/1 0/3(100%) Positive -(100%) (0%) 0/7 (0%)Positive 1/7 4/8(14%) 0/7 (0%) (50%) qPCR 0/8 (0%) 7/77/8 7/7 (100%) (100%) 0/8 ɸ(0%) (88%) 8/87/8 7/7 (100%) (100%) 1/8 (13%)12 (88%) 50Total 0b 10 of a,b,d,e 87Positive a,b,d,e Pathogens 2021,BNZ BNZ 10, 4040 mg/kg/day—20 x FOR PEER REVIEW mg/kg/day—20 days days 8/8 8/8 (100%) (100%) 8/8 7/8 (100%) (88%) 1/1 - 1/7 8/8 (14%) (100%) 7/7 8/8 (100%) (100%) 8/8 8/8 (100%) (100%) 5 0of 0 10 Pathogens BNZ 40 2021,BNZ mg/kg/day—40 10, 40Strain 40 mg/kg/day—20 x FOR PEERdays days days REVIEW 7/8 (88%) Survival 8/8 (100%) 7/7(100%) (100%) Positive 7/8 (88%) - - 1/1 (100%) 0/7**(0%) 1/7 (14%) 7/76/6 (100%) 7/7 (100%) 7/7 Total (100%) 8/8 (100%) Positive Survival 00 b 5 of b 0 10 Positi BNZ BNZ 40 £ mg/kg/day—40 mg/kg/day—40 daysGroup 7/8 (88%) 7/8 (88%) 7/77/7 (100%) § - 6/6 0/7 (100%) (0%) ɸ (100%) 7/7 (100%) 7/7 (100%) 7/7 (100%) 0 BNZ 40 mg/kg/day—40 days Untreated 8/8 (100%) 7/8 (88%) 8/8 (100%) 7/7 (100%) - FBE - 8/8 (100%) 0/7 (0%)8/8 (100%)7/7 (100%) 8/8 (100%)7/7 (100%) 0 0 b Strain (*) £All mice died Positive with Group qPCR FBE; (**) 1 mouse died after presenting positive positive FBE. £ Swiss female mice Strain Group were inoculated with 5 × Untreated BNZ 100 mg/kg/day—20 Animals 8/8 infected (100%) § Up towith 8/8 Y strain0/1 (100%) presented - a survival 8/8 (100%) rate of 63% 8/8 (100%) (100%) when 8/8 (100%)untreated,Cure (100%) §and 000 Mice FBE # 103 trypomastigotes of the Y, anddays Untreated VL-10 strains.7/88/8 (88%) (100%) 6/7 30 8/8 (88%) days(100%) after treatment. (0%)- 5/7 ¶ Fresh 8/8 (71%) blood (100%) Blood 7/7 exam 8/8 (FBE) (100%) after 7/7 Heart Colon cyclophosphamide 8/8 (100%) al VL-10 Positive BNZ 100 100mg/kg/day—40 mg/kg/day—20 ɸUntreated 0%days daysAnimals cure rate, infected Total 7/8Table(88%) Positive 8/8 (100%) with 1. Treatment 6/7 Y FBE (88%)8/8 strain with (100%) presented 0/1 100 (0%)mg/kg/day - 4/8 a 5/7 survival 8/8 of (71%) (100%) rate BNZ 7/7 of 63% improved (100%) 8/8 (100%) when 7/7 the untreated, (100%) survival 8/8 (100%) and rate 0 and 0 VL-10 BNZ immunosuppression. BNZ 100 mg/kg/day—20Quantitative Animals days 8/8 real-time 7/8 infected (100%) polymerase (88%) with 5/8 6/7(63%) chain Y strain ¶ reaction (88%) 0/3 presented (0%) (qPCR) 0/1 (0%) after a (50%) survival cyclophosphamide 5/7 (71%) rate 7/8 (88%) 7/7Colon of 63% when 7/8 immunosuppression. (100%) (88%) 7/7the untreated, (100%) Blood12 a,b,d,e0 10 and ¶ Pathogens VL-10 qPCR 2021, BNZ was 10,100x FOR BNZ PEER mg/kg/day—40 performed 100 REVIEW mg/kg/day—20 5 days 0% to after cure 100%, days with daysAnimals rate, 8/8 immunosuppression, Table a cure (100%) 7/8 infected 1. rate (88%)Treatment of 5/8 only with 75% (63%) 6/7 for forYwith (88%) negative strain the 0/3 100 Blood (0%) FBE presented 20-day 0/1mg/kg/day (0%) assays.4/8 Heart treatment, (50%) a survival 5/7 Tissue of (71%)BNZ qPCR which 7/8 rate improved (88%) was7/7 of 63% increased (100%) performed 7/8 when Mice to (88%) 307/7survival an untreated, 87% (100%) days 12 after5cure rate of a,b,d,e 0 VL-10 BNZ BNZ 40100 mg/kg/day—20 mg/kg/day—40 0% days cure Untreated days rate, 8/8 8/8 Table (100%) (100%) 1. Treatment 8/85/8(100%) Cure 5/8 (63%) # (63%) with0/3 - 100 5/5 (0%) mg/kg/day (100%)8/8 4/8(100%) (50%) *of 8/8BNZ 7/8(100%) improved (88%) - 8/8 7/8 the (100%) (88%) survival - 12 0 rate(100%) 5/5 a,b,d,e immunosuppression BNZ 100for 40 mg/kg/day—20 BNZ all mg/kg/day—40 to 100%, mice. rate Pathogens days 0%days # Cured: when 2021, cure with when the 10,8/8x FOR rate, atreatment (100%)cure all 8/8 PEERTable tests rate (FBE (100%) 8/8 1.and of duration REVIEW Treatment 75% (100%) qPCR) 5/8 for (63%) was the-with were 20-day negative. extended 0/3 100 (0%)8/8 mg/kg/day treatment, Letters to (100%) 40 4/8 represent days. (50%) 8/8 of which a BNZ However, (100%) 7/8 improved increased significant (88%) for 8/8 to7/8 difference this (100%) anthe 87% in strain, (88%) survival cure cure 0the 12 rate a,b,d,e FBE ns 2021, 10, x FOR PEER REVIEW to 100%, with a“b” cure rate of 75% for the- 0/8 20-day 6/6 ** treatment, which 5 increased of 10 to(25%) an 5/8 87% cure rates BNZ BNZ40 when 40mg/kg/day—40 compared to: BNZ mg/kg/day—20 Untreated “a” rate 100 Untreated days mg/kg/day—20 daysto when group; 100%, 7/8 the8/8 5/8 (100%) with (88%)(63%) treatment BNZdays a cure 8/8 100 7/75/5 8/8 (100%) (100%) rate (100%) duration (100%) mg/kg/day—20 of 75% was for - * days; the extended (0%) 8/8 “d” 20-day BNZ to -1/840(13%) (100%) treatment, 40 days.6/6 Untreated 8/8 mg/kg/day—20 (100%) 1/8 (100%) - which However, (13%) 7/7 8/8 days 5/5 increased for 2/8 (100%) and (100%) (100%) this “e” to BNZ strain, an (63%) 0 0 87% 2/8 (25%) 0the cure5/5 (10 ¶ Blood 40 mg/kg/day—40 BNZY40 mg/kg/day—20 Heart days. BNZ lower rate 100 Colon dose when daysthe mg/kg/day—40 of BNZ 8/8 Mice treatment days(40 mg/kg/day) (100%) 8/8(100%) duration 8/8 (100%) was resulted extended 1/8 - (100%) (22%) 6/6to in **0/7 no 8/8 40 cure (100%) days. (0%) for 8/8 either However, 0/8 (100%) (0%) 20-day for 8/8 0/8this orstrain, (100%) (0%) 40-day the 1/8 0 (13%) BNZ BNZ 40100 mg/kg/day—40 mg/kg/day—20 daysdays rate when7/8 8/8 (88%) of (100%) the treatment 7/7 0/8(100%)(0%) duration - 1/8 resulted (13%) was40extended 1/8 BNZ 6/6(13%) ** to 100 6/62/8 (100%) mg/kg/day—20 (25%) 7/7 (100%) days 2/8(100%) (25%) 8/8 750 (100%) a,d,e 0/8 (0 (*) All mice BNZdied 40 mg/kg/day—40 with positive BNZ lower lower 40 days treatments. FBE; dose Animals (**) dose mg/kg/day—20 1 7/8 Moreover, mouse of BNZ (88%) infected BNZ died days (40 7/7 after (40 mg/kg/day) survival with (100%) Y rates strain presenting mg/kg/day) 8/8 (100%) with -resulted presented positive 7/8 (88%) FBE. in (100%) mg/kg/day a £no survival in 1/1 6/6 Swiss no **40 cure cure (100%) 6/6 of rate days. female for (100%) BNZ of for1/7 63% However, either micefor either (14%) 40 when were 20-day 7/7 days 20-day 7/7 fordecreased or untreated, inoculated (100%) this or strain, 40-day 40-day 08/8 (100%) and the Y(*) All BNZ BNZ 100 40 mg/kg/day—40 diedmg/kg/day—40 days treatments. lower days5 of 18/8 dose 10(100%) Moreover, 7/8 (88%) ofdied BNZ 1/8§(40 survival 7/7 mg/kg/day) (22%) (100%) 0/7with (0%)Y40 -resulted0/8 BNZ (100%) (0%) in100 ¶no mg/kg/day—40 0/8cure 6/6 (100%) (0%) for 1/8 either days 7/7 (13%) 20-day 8/8 (100%) (100%) 87 a,b,d,e orfor40-day 0 1/8 (22 %) 5/5 (100%) with (*) All5 ×mice mice died with- positive *103 trypomastigotes BNZ with positive with of 0% 40FBE; the- Y,(**) increase cure and rate, mg/kg/day—40 treatments. FBE; (**) 1 mouse in VL-10 5/5 Table Moreover, mouse treatment (100%)strains. days1. died after Treatment duration survival after Up 7/8 torates presenting 0presenting (88%) 30 (100% with rates Animals days positive 100 7/7 with survival after (100%) infected positive 40 mg/kg/day FBE. treatment. mg/kg/day mg/kg/day with FBE. BNZ £ (100%) Swiss for Yof20 -40 £ Swiss of female days Fresh BNZ strain of BNZ female ofmice blood improved 0/7 BNZ presented mg/kg/day—20 forexam treatment (0%) micefor 40 were 40 werea days the 7/7 days inoculated (FBE) days and decreased survival (100%) survival inoculated 88% after decreased 8/8 rate (100%)rate 7/7 of (100%) 63% 7/8whe (88 BNZ 40 Animals mg/kg/day—20 infecteddays with Untreated treatments. 8/8 Y strain animals (100%) presented infected 7/8 (88%) a survival with 1/1 VL-10 (100%) rate strain of 1/7 63%presented (14%) when 7/7 untreated, similar (100%) features 8/8and (100%) when the 0 fol- %) with1/8 5(*) 0/8 (0%)cyclophosphamide ×(13%) 10 3trypomastigotes All3 mice1/8 died with40 withofdays positive immunosuppression. (13%) to 2/8 increase the (25%) 100%, Y,FBE; of and with in VL-10 (**) treatment).a2/8 1Moreover, ɸ treatment cure strains. mouse Quantitative (25%) rate died of § 75 survival duration Up 75% after a,d,eto for30 ratesafter (100% days presenting real-time the 20-day with survival positive polymerase 40 mg/kg/day treatment. treatment, for FBE. 20£¶Fresh ¶ chain days Swiss which of BNZ blood female reaction treatment exam increased micefor(FBE) (qPCR) 40 were to and an days 88% after inoculated after 87% decreased cure for with 5BNZ × 10 0% trypomastigotes 40 cure mg/kg/day—40rate, with Tableof lowing the days increase 1. Y, and Treatment treatments7/8 in VL-10 (88%) ɸ treatment strains. with were 100 7/7 0% duration § compared: Upcure mg/kg/day (100%) to rate, 30BNZ(100% days -Table of 100 survival after BNZ 1. Treatment mg/kg/day 0/7 BNZ treatment. improved (0%) for 40 20 days with mg/kg/day—40 Fresh for 7/7the 20 (100%) of 100 blood survival days, treatment mg/kg/day exam BNZ days rate 7/7 (FBE) 40 (100%) and of 7/8 88% after mg/kg/day BNZ (88%)0 for b improved 7/7 (10 %) 1/8 (22%) cyclophosphamide with cyclophosphamide 0/7 (0%) 5 × 1030/8 immunosuppression. trypomastigotes 400/8 immunosuppression. (0%) days with (0%)ofofthe Untreated Untreated increase treatment). Y,1/8 Blood and animalsqPCR ɸ (13%)in treatment Quantitative VL-10 was strains. 87 infected performed 8/8 (100%)duration § Up to 30 (100% a,b,d,ereal-time with 5VL-10 days 8/8 polymerase daysafter (100%)strain survival after chain treatment. immunosuppression, presented -days. for 20 days ¶reaction Fresh similar 8/8 (100%) offeatures blood only treatment (qPCR) for 8/8examnegative (100%) after (FBE) when andafter the 88% for 8/8 cyclophosphamide to 100%, with rate immunosuppression. a40 for cure when days 20 of the days, rate of and75%treatment treatment). BNZ Quantitative for 40was ɸ the duration to mg/kg/day 20-day 100%, was real-time with treatment, forextended a 40 polymerase cure days which rate to (0% of4075% chain cure), increased for However, the Table to reaction an 20-day 1. 87% By for (qPCR) cure this treatment, changing strain, after thewhich the(100%) time increase presented %) 7/8 (88%) cyclophosphamide FBE a survival cyclophosphamide assays. 1/1 (100%) Tissue rate of 63% immunosuppression. qPCR 1/7 (14%)when was immunosuppression. 40 performed untreated, 7/7 days Untreated Blood 30 and of days qPCR treatment). animals after infected performed Quantitative immunosuppression with 5 days real-time VL-10 after for all immunosuppression, polymerase strain mice. presented # Cured: chain similar when only reaction all for features tests negative (qPCR) (FBE when and after the cyclophosphamide Untreated BNZ 100(100%) following immunosuppression. lower dose Untreatedtreatments mg/kg/day—20 8/8 8/8BNZ Blood of (100%) qPCR animals (100%) were days (40 was 8/8 7/8 0 (88%) BNZ compared: performed mg/kg/day) infected rate (100%) when with the 6/7 5-VL-10 days resulted100 (88%) treatment mg/kg/day after 8/8 in strain 0/1 no duration (100%) (0%) immunosuppression, cure presented for 8/8 20 for Untreated was 5/7 (100%) days, (71%)BNZ either similar extended only 7/7 20-day features 8/8 40 for to (100%) mg/kg/day (100%) negative 40 or 8/8 when days.40-day (100%) 7/7 0 the (100%) However, 8/8 (10f FBE assays. rate VL-10 Tissue when the of the treatment standard aduration protocol was (BNZ extended 100 mg/kg/day to BNZ 40 days. for However, 20 days)for to this BNZ strain, 100 themg/kg/day for 0/7qPCR was performed 307/7 days after immunosuppression 5for allcompared mice. Cured: when allBNZtests (FBE and # th 1007/7mg/kg/day %) (100%) qPCR) cyclophosphamide were of -BNZ negative. improved Letters (0%) immunosuppression. represent following the 7/7 survival (100%) significant Untreated treatments rate Blood (100%) were qPCR difference animals 0was b in performed infected compared: cure rates with whendays VL-10 100 after mg/kg/day strain immunosuppression, to: “a” presented for 20 Untreated days, similar only group; for“b” features 40 mg/kg/daynegative when the FBE assays. Tissue qPCR BNZ for was 100 20 mg/kg/day—40 days, performed and 30(88%) BNZ days days 40 after 8/8 mg/kg/day (100%) immunosuppression for 5/8 40 (63%) days (0% formg/kg/day allBNZ 0/3 mice. (0%) cure), 100 Table # Cured: 4/8 mg/kg/day—20 (50%) 1. when By all 7/8 changing tests days (88%) (FBE the 7/8and time (88%) 7/8 (88%) qPCR) BNZ BNZ were FBE 100 100 lower assays. mg/kg/day—20 mg/kg/day—20 negative. dose Tissue Letters of qPCR days; treatments. following 40 for BNZ days days, represent was “d” 20 BNZ(40 following days, there a40 performed Moreover, treatments 7/8 is mg/kg/day) significant an mg/kg/day—20 treatments days survival were 30 improvement 6/7 compared: lower (88%) resulted difference after were days in rates dose cure 0/1 in for immunosuppression and compared: no “e” with BNZ of(0%) therapeutic rates BNZ cure BNZ 40 100 when VL-10 BNZ 40 mg/kg/day 5/7 for(40 (71%) efficacy compared for mg/kg/day) either all mg/kg/day—40 100 mice. mg/kg/day of 7/7for (12%). 20-day to: “a” # BNZ20 (100%) Cured:These days. for for days, resulted or20 40 BNZ 7/7 results 40-day Untreated when days, days in (100%)40 group; all BNZ no tests decreased mg/kg/day indicatecure 40“b” (FBE 0 mg/kg/dayand 6/7 (88 for that either 2 the 20-dayVL-10 treatment, qPCR) which were increased negative. Letters BNZ of to40 withthe an 87% represent mg/kg/day—20 standard increase aand cure in BNZ significant protocoldays40 treatment mg/kg/day difference (BNZ 8/8 (100%) duration100 in cure mg/kg/day (100% 8/840 rates days when (100%) for survival (0% compared 20 BNZ cure), for Table to:8/8 - 20mg/kg/day—40 days) 100 to days “a” BNZ of 1. (100%)By Untreated 100 changing treatment 8/8 mg/kg/day days group; (100%) and the 8/8 “b” 88%for time (100%) 8/8 40 for (100%) 5/8 (63 BNZ 100 BNZ 100 mg/kg/day—20 qPCR) treatments. mg/kg/day—40 were negative. days;for Letters Moreover, “d”20 days both days, BNZ represent 8/8 40 strains, survival and (100%)BNZ mg/kg/day—20 aYsignificant andBNZ rates 40 5/8mg/kg/day VL-10, with treatments. (63%) days difference and themg/kg/day 0/3for “e” therapeutic (0%)40 Moreover, BNZ in cure rates days 4/8 40 (0% survival (50%) mg/kg/day—40 efficacy whenfor cure), is 40 7/8 time- compared Table rates (88%) with days. and 1. By40 changing 7/8 mg/kg/day dose-dependent to:decreased “a” (88%) Untreated group; the 12of time in the BNZ a,b,d,e the time “b” for 40 %) 8/8 (100%)to as extended BNZ 40 100 - mg/kg/day—20 days. 8/8 (100%) However, for of8/8 days; the this days, for “d”strain, there 20 standard (100%) BNZ isdays, the 40 an8/8 and protocol (100%) mg/kg/day—20 improvement (BNZ 40 040mg/kg/day 100 days in mg/kg/day and therapeutic “e” for BNZ of 40 BNZ for 40 efficacy days 20(12%). (0% days) mg/kg/day—40 BNZ 40 days cure), to These mg/kg/day—20BNZ Table days. 6/6 1001.mg/kg/day ** results By changing indicate days that forfor 40 8/8 (10 BNZ100 BNZ 40 withmg/kg/day—20 increase BNZ 40 of acute in 40 days days the days; of standard “d” phase, mg/kg/day—40 treatment When treatment). 8/8 BNZ (100%) i.e., duration infected protocol 40 the days with 8/8 mg/kg/day—20 efficacy (100% (BNZ the (100%) with 7/8 100 increase increases (88%) survival VL-10 mg/kg/day days strain, -proportionally and 7/7 for in20(100%) all 8/8 treatment “e” daysBNZ the (100%) formice of 20 days) duration 40treatment 8/8 mg/kg/day—40 -to treatedthe to(100%) BNZ (100% increase and in the 100 days. 88% 8/8 in chronic (100%) mg/kg/day survivaldose 6/6 for for8/8 or (100%) phase 20(100%) length for days with 0 40 7/7 of of treatm (100%) %) 6/7 (88%) y) resulted in no0/1cure (0%)for 5/7 (71%)20-day either 7/7 (100%) days, of there or the 40-dayis 7/7improvement standard an (100%) protocol 0 (BNZ in 100 therapeutic mg/kg/dayefficacy for (12%). 20 days) These to (100%) BNZ results 100 indicatemg/kg/day that for for 40 both days, treatment.strains, Untreated When there is Yan infected and animals VL-10, with improvement the40 the infected VL-10 days therapeutic in with strain, therapeutic of VL-10 treatment). efficacy all strain the6/6 efficacy mice is ** time- presented treated (12%). and These in dose-dependent similar the features chronic results indicatephasewhen inwith that thethe for %) s with 5/840(63%) mg/kg/day 0/3BNZ(0%) 40 of40BNZ days(*)forof mg/kg/day—40 4/8 (50%) All40 treatment). 100 miceboth days7/8 mg/kg/day days died (88%) When days,withthere strains, decreased 7/8Y7/8 infected positive for (88%) and 40 is(88%) an days with VL-10, FBE; 7/7 (**)the improvement survived (100%) 121the a,b,d,e VL-10 mouse and instrain, therapeutic died 62% - afterall therapeutic were the efficacy presenting cured, BNZmice efficacy 40 istotime- Table mg/kg/day—40 6/6 treated (12%). positive (100%) and 2, although inThese FBE. the 7/7days chronic results dose-dependent £ Swiss when (100%) female 7/8 phase treated indicate mice (88%) in with 0 thatinoculat the were 7/7 (10 for acute following 100 both phase, mg/kg/day strains, When When i.e., treatmentsY infected forthe infected and 40 efficacy were days VL-10, with with increases compared: survived the the the Untreated VL-10 VL-10 and therapeutic proportionally BNZ strain, strain, 62% 100 animals allwere (100%) efficacy theall mg/kg/day the cured, infected mice is the mice for Table time- treated increase treated with 20 and 2, indays, inchronic in although dose the BNZ dose-dependent VL-10 the strain orpresented chronic 40 when length mg/kg/day phase phase treated in with of the with similar fe %) 8/8survival (100%) for 20 - days8/8 Untreated (100%) with with 3 animals 8/8 100 5 × treatment. 10 40 (100%) mg/kg/day both trypomastigotes infected mg/kg/day strains, 8/8for (100%) ofY with for 40 and the 40Y,VL-10 days days, VL-10, and 0VL-10 survived strain these and thestrains. presented animals therapeutic62% § Up had weretosimilar already cured, efficacy 30 features shown Table is time- when 2,cure inof although and the ¶25%, orwhich when dose-dependent treatedis ain (FBE) theafteraf (100% (*) All mice diedofwithtreatment acute positive and FBE;phase, 88% (**) i.e., for 1andmouse the efficacy died after increases presenting proportionally positive (*)hadAll FBE. mice £days todied Swiss after the with female treatment. increase positive mice dose were Fresh FBE; (**) blood length inoculated1whenmouse exam of died following 6/6 **treatments for with acute 100 20100 40 days,mg/kg/day mg/kg/day phase, mg/kg/day i.e., BNZ the for forfor 40 40 40 40 efficacy days days mg/kg/day days, following survived these increases survived for animals treatments and 40and days proportionally 62% 62% (0% were were were alreadycure), cured,to compared:cured, the Table shown Table Table increase BNZ 2,1. cure By2, 100in although although changing of 25%, dose mg/kg/day which or when the length time for treated is treated a of 20 days, BN cyclophosphamidepromising with treatment. 40were acute result mg/kg/day compared: phase, considering immunosuppression. i.e.,for the BNZ 40 days, efficacy 100resistance the ɸ mg/kg/day theseQuantitative increases animals for the with had20 ofproportionally5 ×days, evaluated real-time already BNZ strain. shown to 103 trypomastigotes 40 ¶ polymerase mg/kg/day theblood All cure ofof increase the chain ofthe mice 25%, in dose Y, infected reaction which and or VL-10 is(qPCR) length a of af 40astrains. aBy cU § %) 7/7 (100%)with 5 -× 103 trypomastigotes 6/6 of of the promising treatment. with the (100%) with 40 Y, standard and 40 result mg/kg/day 7/7 VL-10 mg/kg/day (100%) protocol strains. considering for for (BNZ 40 for § Up to 020days, 40 the days, 100 30 resistance days, these days these mg/kg/dayand animals after animals treatment. for ofperformed BNZ the 40 had had 20 evaluated days) mg/kg/day already Fresh already to strain. shown BNZ forshownAll 40 100 cure exam of dayscure (FBE) mg/kg/day the25%, of (0% of mice 25%, after cure), which which for infected Table is is1.negati th VL-10 strain presented for 20 days, (100%) similar and cyclophosphamidewith BNZ promising features the 40 VL-10 mg/kg/day strain immunosuppression. treatment. when result and ɸ considering for 40 untreated days Blood (0% survived, qPCR the real-time resistance cure), was of Table but the no 1. evaluated cyclophosphamide By cure 5 dayschanging was afterobserved. strain. the time immunosuppression, All of(qPCR) immunosuppression. the mice infectedɸonly for Quantitativ cyclophosphamide Table 1. Time- and immunosuppression. dose-dependence days, with promising there the of promising BNZ VL-10 isthe result an treatment result Quantitative improvement strain and considering onuntreated considering different the ofdifferent the in Trypanosoma the therapeutic resistance survived, resistance standard polymerase cruzi ofefficacy protocol but the of nothestrains evaluated (BNZ (12%). cure chain evaluatedin was 100 These strain. reaction theobserved. acute mg/kg/day Allphase strain. results ofAll of indicate the for Chagas ofmicethe after mice that infected infected for FBE assays. Tissue theqPCR was performed 30 days after immunosuppression for all mice. of20 days) to BNZ(FBE 100apm # Cured: when all tests e died after presenting d: BNZ 100 Table 1. Time- cyclophosphamide mg/kg/day disease. for of20 positive the and standard FBE. days, £ Swiss dose-dependence with immunosuppression. BNZ 40protocol female mg/kg/day mice ofVL-10(BNZ BNZ were Blood 100 strain qPCR mg/kg/day inoculated treatment and on was untreated performed for 20 5days) Trypanosoma survived, to after BNZ dayscyclophosphamide cruzi but no 100 curemg/kg/day strains immunosuppression, in was immunosuppression. the acute observed. for only40 phase for Chagas negative Blood qPCR was Table 0 strains. § Updisease. to 30 1. Time- days after days,and dose-dependence qPCR) treatment. there were is both ¶with an with strains, the negative. Fresh blood improvement ofthe VL-10 BNZ Y LettersVL-10 exam and treatment strain in strain VL-10, represent (FBE) and therapeutic on after and a the untreated different untreated days, therapeutic there significant efficacy Trypanosoma survived, is an survived, (12%). efficacy but improvement difference FBE These cruzi in no cure assays. but is no time- strains cure rates results in Tissue cure in was and the therapeutic when indicate qPCR was acute observed. observed. dose-dependent compared that was phase efficacy for to: performed of“a” Chagas (12%). in Untreated the These days after imm“ result group; for 40 days (0% FBEcure),TableTable assays. 1.Tissue Time- and 1. qPCR By100 dose-dependence was performed changing the time of days 30 BNZ after treatment on different Trypanosoma immunosuppression for all cruzi mice. # strains Cured: in the when acute all tests phase(FBEof30 Chagas and Quantitative disease. real-time qPCR) disease. were BNZ polymerase both strains, negative. acute mg/kg/day—20 chain Y and phase, reaction VL-10, i.e., days; the (qPCR) “d” the therapeutic efficacy BNZ afterboth 40 increases mg/kg/day—20 strains, efficacy in cure is proportionally Y and days Positive time- qPCR)VL-10, and andqPCR were to “e” the the BNZ dose-dependent increase 40 therapeutic negative. Letters in thegroup; “b” of in mg/kg/day—40 dose efficacy represent or is length days. a time- significant and differen dose-d mg/kg/day forTable qPCR was performed 20 days) 2.5Time- days to BNZ 100Letters mg/kg/day and dose-dependence after represent treatment. immunosuppression, for of BNZ a 40significant only treatment for difference negative on VL-10 Trypanosoma rates when Positivecruzi compared strain qPCR to: “a” Untreated in the chronic phase of Chagas BNZ 100 mg/kg/day—20 acute phase, i.e., the days; “d”efficacy BNZ increases 40 mg/kg/day—20 Survival acute proportionally Positive phase, days and “e” i.e., toBNZthe theBNZ efficacy 100 increase 40ɸmg/kg/day—40 Positive qPCR increases mg/kg/day—20 in dose or proportionally days. days; length “d” of Total Positive BNZ to40 the increase mg/kg/day—20 in rapeutic ys efficacydisease. after immunosuppression (12%). These for all results mice. #indicate Cured: when that for all tests When (FBE and infected with the VL-10 Positive strain, qPCR all the mice treated in the chronic pha treatment. Survival treatment. Positive ɸ Total Positive apeutic cant efficacy difference Strain in £ cure Table is 1. time- rates Time-whenand Group and dose-dependent compared to: “a” of dose-dependence inBNZ the treatment Survival Untreated group; Positive “b” on different for 40Trypanosoma cruziɸ and strains in the acute Total phase Positive of Chagas Cure # When § 100 infected Survival mg/kg/day with the Positive VL-10 days survived strain, all the mice 62% treated were in cured, the chronic Table When Total 2, Positive although infected ## with the when Strain £ Group Positive ɸ Total phase Cure Cure with es proportionally kg/day—20 Strain days to the and “e” BNZ 40 disease. increase Group in mg/kg/day—40 dose Table or length days. 1. Time- of and § with 40 dose-dependenceFBE mg/kg/day offor BNZ 40 Positive days, treatment qPCR these on animals different had Trypanosomaalready shown cruzi 100 mg/kg/day strains cure for 40 dayswh in of the 25%, acute ph su Table 1. Time- Strain £ and dose-dependence Group of BNZ100 treatment Survival mg/kg/day on different§for 40 Trypanosoma days ¶ survived cruziand strains62%inwere the acute cured, phase Table of Chagas 2, although when treated Cure # Strain £ Groupdisease. FBEFBE Blood Blood Heart Heart ColonColon Mice Positive mice (%) Cure # disease. with 40 mg/kg/day promising § for 40 result these FBE days, considering animals the had Positive resistance already qPCR Colon ofshownthe evaluated with cure of 40 strain. 25%, mg/kg/day which All of is the for a mice 40 day i d with the VL-10 strain, allUntreated the mice treated in 8/8the (100%) chronic phase 5/8 (63%) ¶ FBE Blood ¶ with 2/3Blood (67%) Heart 4/5 * (80%) 2/5 (40%) Mice 8/8 (100%) 0 with the VL-10 strain and untreated Heart survived, Colon but no cure promisingMice was observed. result considering Trypanosoma cruzi strains BNZ 100 mg/kg/day—20 days in the acutecured, Untreated phasedays promising of Chagas 8/8 5/8result (100%) (63%) considering 5/8 (63%) 5/5Positive (100%) the resistance ¶ Positive 2/3 (67%) *qPCR Blood of the- evaluated 2/7 * (29%) Heart 1/8strain.4/7 (57%) - (13%) Colon All of(100%) 8/8 Positive Total 5/5 the mice (100%) qPCRinfected Mice 0 40 days survived VL-10 andBNZ62% 100 were mg/kg/day—40 Table 2, Survival 8/8although (100%) when2/8 treated (25%) 0/6 (0%) 0/8 ɸ (0%) 3/8Positive (38%) 0 a,b Untreated with the VL-10 5/8 (63%) strain and 5/5 untreated (100%) survived, * but - no cure was - observed. with 5/5 the VL-10620strain (100%) and un 75 0a,b,c,d c BNZ BNZ 40 mg/kg/day—20 100 mg/kg/day—20 days days 8/8 8/8(100%)(100%) 4/80/8(50%)(0%) 3/4 (75%) 1/7 * (14%) 1/7 (14%) 8/8 (100%) y for 40 days, these Strain £ BNZ animals had Untreated BNZ 40 mg/kg/day—40 Group already Survival days shown cure 5/8 8/8Positive (100%) of 25%,6/8 (63%) which 5/5(75%)(100%) is a 1/8 0/2 (13%) Survival ɸ (0%) * 1/8 (13%) 0/5 *Positive -(0%) Total 2/8 Positive (25%) 0/5-(0%) 2/8 5/5 (25%) 6/8(100%) ɸ (75%) Cure 25 a,d,e 0 # Tota Y BNZ 100mg/kg/day—40 100 mg/kg/day—20Untreated days 8/8 days 8/8 (100%) (100%) § 5/8 (63%) 0/8(22%) 1/8 (0%) 5/5 (100%) 1/8 0/7 (13%) (0%) * 1/80/8 (13%) (0%) - 2/8 0/8 (25%) (0%) - 1/8 2/8 (13%) (25%) 5/5 (100%) 8775a,b,d,e a,d,e 0 onsidering the Positive resistance qPCR BNZ 100 of the evaluated mg/kg/day—20 strain. £days All of 8/8 (100%) the mice infected 0/8FBE (0%) with 1/8 (13%) × 10 1/8 (13%) 3 trypomastigotes 2/8of(25%) 2/8 (25%) 75 a,d,e n£ Y (*)GroupMice BNZ BNZ died 100 BNZ 40 with 100positive mg/kg/day—40 FBE. days Strain mg/kg/day—20 mg/kg/day—20 Swiss daysfemale 8/8 (100%) (100%) mice 8/8Group were (100%) 1/8inoculated (22%) 0/8 (0%) 0/7(100%) 51/8 (0%) § (13%) 0/8(14%)(0%) 1/8 (13%) 0/8(100%) the (0%)2/8 VL-10 (25%) strains 1/8 Cure (13%) # 2/8 and(25%)were87 0a,b,d,e 75 a,d,e ain and untreated Ytreated survived, BNZ 120 days100 after no cure§days butinfection. mg/kg/day—40 was Up days 8/8 toobserved. 30 days 8/8 after(100%) treatment. 7/8 1/8(88%) ¶ Fresh 1/1 (22%) blood0/7 Blood (0%) exam 1/7 (FBE)0/8 Heart after(0%) 7/7 0/8 Colon cyclophosphamide FBE 8/8 (0%) immunosuppression. 1/8(100%) Mice(13%) 87 a,b,d,e a,b,d,e Y BNZ BNZ 40 BNZ mg/kg/day—20 100 mg/kg/day—40 Total days Positive days 8/8 FBE (100%)8/8 (100%) 7/8 (88%) 1/8 (22%) 1/1 (100%) 0/7 (0%) 1/7 (14%) 0/8 (0%) 7/7 (100%) 0/8 (0%) 8/8 (100%)1/8 (13%) 0 87 BNZ4040mg/kg/day—40 days chain 7/8 8/8(88%) 7/77/8(100%) 1/1 -(100%) immunosuppression. 0/7 1/7(0%) 7/77/7(100%) 7/78/8(100%) 0 0 b ɸ Quantitative mg/kg/day—20 real-time polymerase days reaction (100%) (qPCR) after (88%) cyclophosphamide (14%) (100%) Blood qPCR Blood (100%) was Heart performed 0Colon ¶ 5 daysBNZ after 40BNZ mg/kg/day—40 40 mg/kg/day—20 immunosuppression, days only days for 7/8¶ (88%) negative 8/8FBE (100%) Blood 7/7 (100%) assays. 7/8 (88%) Tissue Heart-was qPCR 1/1 (100%) Colon 0/7 (0%) performed 1/7 30 (14%) days Mice 7/7after (100%) 7/7 (100%) 7/7 (100%) immunosuppression 8/8 (100%) for all b 0 BNZ 40 mg/kg/day—40 Untreated days Cure 7/8 5/8 # (88%) (63%) 7/7 5/5 (100%) (100%) *- 0/7 -(0%) 7/7 (100%) - 7/7 5/5 (100%) (100%) 0b mice. # Cured: BNZwhen 40 mg/kg/day—40 all Untreated tests (FBE and days qPCR) were 7/8 (88%) negative. 8/8 (100%) 8/8 (100%) Letters 7/7 (100%) represent -a - significant 0/7 difference 8/8 (100%) 8/8 (100%) (0%)in cure 7/7 rates (100%) when compared 8/8 (100%) 7/7 (100%) to: “a” 0 0 b Untreated Untreated BNZgroup; 100 mg/kg/day—20 “b” BNZ 100 Untreated 5/8 (63%) days 5/5 mg/kg/day—20 8/8 8/8 (100%) (100%) Untreated days; (100%) “c” BNZ 8/80/8 * (0%) 100 (100%) mg/kg/day—401/8- 5/8(13%) - (63%) days;1/8 8/8 - “d”(13%) 5/5 (100%) (100%) BNZ 40 5/5 2/8 8/8 (25%) mg/kg/day—20 (100%) (100%) * 8/82/8 0 (25%) days. (100%) - 750a,d,e - 5/5 Blood Heart BNZ 100 Colon mg/kg/day—20 UntreatedMicedays 7/88/8(88%) (100%) 6/7 8/8 (88%) (100%) 0/1 (0%) - (100%)5/7 8/8 (71%) (100%) 7/78/8 (100%) (100%) 7/7 8/8 (100%) (100%) 0a,b,d,e 0(25%) 100Ymg/kg/day—20 BNZ VL-10 BNZ 100 BNZ 100 mg/kg/day—20 mg/kg/day—40 days 8/8 Untreated (100%) days days BNZ0/8 8/8 100 7/8 (100%) mg/kg/day—20 (0%) (88%)8/8 1/8 (100%) 1/8 (13%) 6/7 (22%) (88%) days 8/8 1/8 (100%) 0/7 8/8 (13%) 0/1 (0%) (0%) 2/8 - 0/8 (25%) 5/7 (0%) 0/8 (71%) 8/8 (0%) (100%) 2/8 7/70/8(100%) (0%) 1/8 (25%)8/8 (13%) (100%) 75 7/71/81/8 (13%) a,d,e (100%)(13%) 8/8 (100%) 872/80 0 2/ VL-10 BNZ BNZ100 mg/kg/day—40 days 8/87/8 (100%) (88%) 5/8 6/7(63%) 0/3 (0%) 4/85/7(50%) 7/8 7/7(88%) 7/8 (88%) 12 0 a,b,d,e 100 mg/kg/day—20 40 mg/kg/day—20 Y(100%) days days BNZ 8/8 100 (100%) mg/kg/day—40 7/8 (88%) (88%) days 1/10/18/8 (0%) (100%) (100%) 1/7 (71%) (14%) 1/8 (22%) 7/7 (100%) (100%) 0/7 (0%) 7/7 8/8 (100%) (100%) 0/8 (0%) 0/8 0 (0%)0 1/ BNZ 100 VL-10mg/kg/day—40 BNZ BNZ 100 BNZ days mg/kg/day—40 100 8/8 mg/kg/day—20 days When 1/8 days 8/8 (22%) (100%) comparing 7/8 0/7 (88%) 5/8(0%) the (63%) 6/7 0/8 (88%) different0/3 (0%) 0/3 (0%) strains 0/1 0/8 (0%) - (0%) submitted 4/8 (0%)(50%) 5/7 (71%) to1/8 7/8the(13%) (88%) 7/7 (100%) same treatment 87 7/8a,b,d,e (88%) 7/7 (100%) during 12 a,b,d,e the * - VL-10 BNZ40 mg/kg/day—20 40-mg/kg/day—20 100 mg/kg/day—40 mg/kg/day—40 5/5 (100%) days days days 8/808/8 7/8 (100%) (100%) (88%) 1/18/8 8/85/8 7/7 (100%) (63%) (100%) 8/84/8 (100%) (50%) 8/87/8 (100%) (88%) 8/87/8 (100%) (88%) 1200a,b,d,e -- susceptibility 0/7(100%) (0%) 7/7(100%) (100%) 7/7 (100%) b BNZ 40 mg/kg/day—20 BNZ BNZ 40 days 8/8 (100%) 100 mg/kg/day—40 days BNZ 7/8 days 8/840(88%)mg/kg/day—20 (100%)8/8 (100%) (100%) (100%) days 5/8 1/7 (14%) (63%) 8/8 (100%) 0/3 7/7 (0%) (100%) 8/8 7/8 4/8 (88%) (50%)8/8 8/8 1/1 (100%) (100%) 7/8 (88%) 8/8 01/7 (100%)(14%) 7/8in(88%) 7/7 0 (100%)12 a,b,d,e 8/8 8 (13%) 1/8 (13%)BNZ BNZ 2/8 (25%) 40 mg/kg/day—20 2/8 (25%)acute daysBNZ phase757/8 of a,d,e 8/8 infection, (100%) 7/7 the 8/8(100%) (100%) profile of 6/6 ** to benznidazole found the litera- BNZ 40 mg/kg/day—40 40 BNZmg/kg/day—40 days 7/8 ture 40 mg/kg/day—20 days (88%) 7/7 days 40 (88%) mg/kg/day—40 (100%) 8/8 (100%) - days 8/8 0/7 (100%) (0%) - - (88%) 7/8 7/7 - 8/8 7/7 (100%) 6/6 (100%) ** 8/8 (100%) (100%) 6/6 7/7 8/8(100%) (100%) (100%)8/8 -(100%) 7/7 8/8b(100%) 0 (100%) 0/7 (0%) 8/8 (100%) 7/7 0 0(100%) 0 7/7 /7 (0%) 0/8 (0%) BNZ0/8 40(0%)mg/kg/day—40 Untreated 1/8 (13%) days [23]87 is8/8maintained: 7/8 a,b,d,e (88%) (100%) 7/7animals 8/8 (100%) (100%) infected -- with8/8 the (100%) 6/6 Y ** (100%) strain6/6 presented 8/8 (100%) (100%) higher 7/7 8/8 (100%) percentages (100%) 00 of BNZ 40 mg/kg/day—40 cure, days FBE;and (**) those 7/8 (88%) infected 7/7 with(100%) the VL-10 - strain (100%) showed 6/6 ** less 6/6 (100%) therapeutic 7/7 (100%) success (Figure 0 2). 0 1 (100%) 1/7(*) All mice died with positive 10mouse 7/8died after presenting positive FBE. £ Swiss female mice were inoculated (14%) BNZ 7/7100 (100%) BNZ mg/kg/day—20 8/8 (100%) 40 mg/kg/day—40 days days 7/8 (88%) (88%) 6/7 7/7 (100%) - 5/7 (100%) 6/6-(100%) 7/7 (100%) VL-10 Untreated (*) with All5 mice × 10 died with positive 3 trypomastigotes 8/8 (100%) of FBE; the Y, 8/8 (**)and 1b(100%) mouse VL-10 Untreated died strains. - §(88%) after Uppresenting to 0/1 8/830(100%) 8/8(0%) days (100%) positive 8/8treatment. after (100%) FBE. (71%) 8/8 (100%) £ (100%) Swiss ¶8/8 7/7(100%) (100%) female Fresh blood mice 7/7 were exam 8/8(100%) 0 (100%) inoculated (FBE) after 8/80(100%) 8/8 - 0/7 (0%) (*) All mice 7/73100(100%) died with 7/7 (100%) positive FBE; (**)08/81 mouse died5/8 after presenting positive FBE. £ Swiss female mice were inoculated a,b,d,e with 5BNZ BNZ 100 cyclophosphamide mg/kg/day—20 × 10 mg/kg/day—40 days trypomastigotes (*) All 3mice died with7/8 (88%) of days the BNZ Y, 6/7 and100 positive FBE; (**) 1 mouse immunosuppression. VL-10 (100%) mg/kg/day—20 (88%) VL-10ɸ 0/1 strains. Quantitative (0%) (63%) § Up died§ afterdays to5/7 30 0/3 (71%)7/8 days presenting real-time (0%) (88%)7/7 after 4/8 (100%) (50%) 6/7 treatment. positive FBE. polymerase (88%) 7/7 ¶ chain £ 7/8 (88%) 0/1 (100%) Fresh Swiss (0%) blood female mice reaction exam7/8 05/7 (qPCR) (88%) (71%) (FBE) were after after 12 7/7 inoculated (100%) 7/7 0 with 5BNZ × 1040trypomastigotes mg/kg/day—20 of daysthe BNZ Y, and8/8 100 VL-10 (100%) ɸ mg/kg/day—40 strains. 8/8 (100%) Up days to 30 8/8days- after8/8 (100%) treatment. (100%) 5/8 (63%) ¶ Fresh 8/8 (100%) 0/3 blood (0%) exam 8/8 4/8 (FBE) after (100%) (50%) 7/8 0 (88%) 7/ cyclophosphamide BNZ 100 cyclophosphamide mg/kg/day—40 days with 5 × 10 trypomastigotes 3 immunosuppression. 8/8 (100%) immunosuppression. 5/8 of theBlood (63%) Y, and Quantitative ɸ qPCRVL-10 0/3 was(0%) strains. performed real-time 4/8 § (50%) Up to5 days polymerase 30 days 7/8 after (88%) after chain7/8 (88%) treatment. Fresh only immunosuppression, ¶reaction 12 (qPCR) a,b,d,e bloodfor examnegative after (FBE) after - cyclophosphamide 8/8 (100%) 8/8 (100%) immunosuppression. 8/8 (100%) BNZ 040 mg/kg/day—20 Quantitative ɸ days real-time8/8 (100%)polymerase 6/68/8 **(100%) chain reaction - (qPCR) 8/8 (100%) after8/8 (100%) 8/8 cyclophosphamide BNZ 40 mg/kg/day—20 FBE cyclophosphamide assays. BNZ Tissuedays immunosuppression. qPCR 8/8 immunosuppression. was(100%) days 8/807/8 performed Blood 30(100%)days qPCR after7/7 was performed Quantitative - (100%)8/8 (100%) immunosuppression 5 days real-time 8/8after (100%) forafter immunosuppression, polymerase all mice. # 8/8 chain 6/6(100%) Cured: when allonly only reaction for 0 (100%) tests negative (qPCR) (FBE and 0 after /1 (0%) 5/7 FBE (71%) assays. 7/740 cyclophosphamide mg/kg/day—40 (100%) Tissue immunosuppression. qPCR 7/7was (100%) performed (88%)qPCR Blood 30 days after was performed -5 days immunosuppression (100%) immunosuppression, (100%) 7/7 for 6/6 negative ** for“b” qPCR) cyclophosphamide were negative. Lettersimmunosuppression. represent a significant Blood qPCR difference was in performed 6/6 cure ** rates 5for whendays all after compared # Cured: when all mice.immunosuppression, to: “a” Untreated tests only(FBE group; andnegative /3 (0%)BNZ 40 FBE 4/8mg/kg/day—40 (50%) assays. 7/8 Tissue days (88%) qPCR 7/8 was 7/8 (88%) (88%) BNZ performed 7/7 12 40 30mg/kg/day—40 (100%) a,b,d,e daysdied afterafter -after days immunosuppression 7/8 (88%) 6/6 for(100%) all7/7mice. £(100%) # 7/7Cured: (100%) - when all 0tests (FBE and 6/6 (100%) 7/7 (*) qPCR) BNZ All mice were FBE 100 died assays. mg/kg/day—20 with negative.Tissue positive Letters qPCR days; FBE; represent wasBNZ “d” (**) performed1significant a40 mouse 30 days mg/kg/day—20 difference presenting inand cure immunosuppression days (100%) “e” positive ratesBNZ when 40 FBE. compared for Swiss all mg/kg/day—40 mice. female to: # “a” Cured: days. mice Untreated whenwere inoculated group; (100%) all tests “b” (FBE and 20 days; “c” BNZ 100 mg/kg/day—40 days; “d” BNZ 40 mg/kg/day—20 days. When comparing the different strains submitted to the same treatment during the acute phase Whenofcomparing infection, the theprofile of susceptibility different to benznidazole strains submitted to the samefound in the during treatment literature the [23] is maintained: acute animals phase of infection, theinfected profile ofwith the Y strain susceptibility presented higher to benznidazole foundpercentages of in the literature Pathogens 2021, 10, 729 cure, [23] and those infected is maintained: with the animals VL-10with infected strain showed the lesspresented Y strain therapeutic success higher (Figure62). percentages ofof 10 cure, and those infected with the VL-10 strain showed less therapeutic success (Figure 2). 87 75 87 75 62 62 25 a 12 25 a a 0 0 0 0 0 12 0 0 0 0 0 a 0 0 0 0 0 0 0 0 0 0 Figure Figure 2. 2. Efficacy Efficacy ofof treatment treatment compared compared between between strains strains and and stages stages ofof infection. infection. “a” “a” represents represents significant significant difference difference inin relation to Acute phase Y. “ “ represents a significant difference among the different phases of infection, but for the [ [ relation2.toEfficacy Figure Acute phase Y. “ of treatment ” represents compared a significant between difference strains among and stages the different of infection. phases of infection, “a” represents butdifference significant for the same in same treatment treatment relation to and and Acute strain strain (p