Review on Experimental Treatment Strategies Against Trypanosoma cruzi

Figures & data

Figure 1 Chemical structures of the most promising nitro-heterocyclic compounds that induce cure with parasite elimination in mice.

Table 1 Classes of Promising New Chemical Entities for Experimental Treatment of Chagas Disease

Mechanism/Target	Chemical Class/Compound	Outcome	Ref.
DNA and kinetoplast-DNA targeting and topoisomerase inhibitors and unknow targets	Amide-containing thiazoles	Safe compounds that had in vitro and in vivo anti-T. cruzi activities.	[^Citation93]
	Arylimidamide derivatives	18SAB075 exhibited best selective index; and slightly reduced parasitemia and mortality in mice infection.	[^Citation92]
	Amidines and analogues Bis-arylimidamides	Bis-arylimidamides displayed in vitro activity. 28SMB032 reduced parasitemia, but without mortality protection. DB1957, DB1959 and DB1890B reduced parasitemia levels and/or mortality.	[^Citation91,Citation94]
	DB1831 and its mesylate salt derivative (DB1965)	Compounds presented in vitro and in vivo activity. DB1965 produced no parasitological cure, but reduced parasite burden and protected against mortality	[^Citation90]
	DB766 and analogues	DB766 displayed potent in vitro activity. In murine infection, the drug reduced parasite load in blood and heart; reduced hepatic and cardiac lesions; prevented electrocardiographic alterations and prevented mortality.	[^Citation87^–^Citation89]
Antimicrobial peptides	AS-48 Bacteriocin	AS-48 was active in vitro and in acute stage of mice infection it reduced parasitemia and in chronic stage the parasitic load.	[^Citation98,Citation99]
Copper complexes	Ternary copper (II) complexes	Compounds with higher selectivity index were able to reduce parasitemia.	[^Citation100]
Cysteine protease inhibitors and Cruzipain inhibitors	Fluoromethyl ketone–derivatized pseudopeptides	K777 is an irreversible peptidyl inhibitor of cruzipain and it was able to reduce parasitemia and mortality, and induce cure in murine model. In infected dogs, it reduced cardiac damage, but without cure. Nonpeptidic tetrafluorophenoxymethyl ketone reduce parasitemia in acutely infected mice with no apparent toxicity.	[^Citation77^–^Citation79] [^Citation81]
	Reversible cruzipain inhibitors containing a nitrile “warhead”	Cz007 and Cz008 presented potent in vitro activity and displayed parasitemia reduction and cure in mice acutely infected.	[^Citation80]
	Thiazole compounds	Thiazole compounds showed in vitro activity and promoted reduction of parasitemia and mortality in infected mice. These compounds inhibited cruzipain.	[^Citation82^–^Citation85]
Ergosterol biosynthesis inhibitors	4-aminopyridyl-based lead compounds	Compounds showed anti-T. cruzi effects in acute and chronically infected mice, but without cure.	[^Citation67]
	Fenarimol analogues	Fenarimol analogues were active in vitro and in vivo against T. cruzi infected mice	[^Citation57,Citation63,Citation64]
	Non-azole LP-10	LP-10 was able to reduce parasitemia and to induce cure in infected mice.	[^Citation65]
	Ravuconazole and E1224 (fosravuconazole)	Ravuconazole presented effective control of infection in mice and dogs. E1224 or fosravuconazole, a prodrug of ravuconazole were well tolerated and effective in suppressing parasitemia. Cure was detected in mice infected by Y strain, but no cure was observed in Colombian infection in mice.	[^Citation116,Citation117] [^Citation118]
	VFV	VFV was more potent than VNI in reducing parasitemia and presented effects on mortality protection.	[^Citation60,Citation119]
	VNI	VNI presented promising anti-T. cruzi activity in vitro and in vivo. The drug promoted reduction of parasitemia in mice infected by different strains. Cure was detected in animals acute and chronically infected by Tulahuen strain.	[^Citation59^–^Citation62]
	VT-1161	VT-1161 is a potent T. cruzi CYP51 inhibitor and revealed in vitro and in vivo activity, as well as suppression of parasitemia peak in mice infection by Y strain.	[^Citation66]
Nitro-heterocyclic compounds and ROS inducers	5-Nitroindazole derivatives	Megazol, a nitroimidazole-thiadiazole derivative, demonstrated a curative action in infected mice. The mechanism of action is related to the impairment of protein synthesis. Megazol derivatives showed in vitro activity. In infected mice, S2 led to parasitemia reduction, and S3 to mortality reduction.	[^Citation50,Citation53] [^Citation54]
	Nitrotriazoles	Nitrotriazole-based compounds exhibited in vitro and in vivo anti-T.cruzi activity. Some compounds are potential inhibitors of T. cruzi CYP51 and substrates for the type I nitroreductase. Triazole-based analogues of BNZ showed in vitro activity. In infected mice, the analogues exhibited lower potency and higher toxic than BNZ.	[^Citation68^–^Citation76] [^Citation58]
	Fexinidazole	Fexinidazole was more effective than BNZ, with cure rate greater than 70% in animals infected by different strains and treated during the acute or chronic phases.	[^Citation46^–^Citation48]
	Fexinidazole metabolites	Fexinidazole sulfone and fexinidazole sulfoxide induced higher cure rates than fexinidazole.	[^Citation47,Citation49]
	Imidazole Derivatives	Phthalazine derivatives containing imidazole rings showed in vitro and in vivo anti-T. cruzi activity.	[^Citation55]
	MK-436 (2,5-nitroimidazole)	MK-436 was highly efficacious against acute and chronic infection by different strains, and induced cure in acute stage.	[^Citation51,Citation52]
	Ruthenium complexes	The ruthenium compounds exhibited potent in vitro and in vivo trypanocidal activities by acting on active cysteine (cys166) site or releasing nitric oxide. NO donor compound led to parasitological cure.	[^Citation101^–^Citation104]
Purine Salvage Pathway	Nucleoside derivatives	Nucleoside analogues presented in vitro activity and the most potent showed suppression of parasitemia and mortality in acutely infected mice.	[^Citation106,Citation107]
	Xanthine analogs	Xanthine analogs showed in vitro activity and were able to reduce parasitemia in CL-infected mice.	[^Citation105]
Oxamates	Benzyl ester of N-propyl oxamate; ethyl esters of N-propyl and N-isopropyl oxamates; and N-isopropyl oxamate	Compounds exhibited trypanocidal activity in vitro and in vivo. They acted as possible inhibitors of T. cruzi α-hydroxy acid dehydrogenase (HADH)-isozyme II	[^Citation108^–^Citation110]
Oxaboroles multiple cellular targets	Oxaboroles	AN4169 showed broad efficacy in vitro against strains of T. cruzi and was a fast-acting trypanocidal. The drug induced cure in mouse model.	[^Citation56,Citation57]
Proteasome inhibitor	Triazolopyrimidine	GNF6702 cure mice in chronic phase of infection with T. cruzi.	[^Citation114,Citation115]
Quinolines	Quinolines	Tested quinolines were more active in vitro than BNZ. DB2186 reached 70% reduction of the parasitemia load in mice infected by Y strain.	[^Citation111]
Sphingosine kinase inhibitor	N,N-dimethylsphingosine (DMS)	DMS blocked sphingosine-1-phosphate production, a cell mediator during inflammatory responses, and exhibited anti-parasitic activity in vitro and in vivo and immunomodulatory actions in chronically infected mice.	[^Citation112]
Squaramides	Squaramides	New long-chain squaramides displayed in vitro and in vivo activity with low toxicity	[^Citation55,Citation113]
Terpene and terpenoid derivatives	Terpenoid derivates	Synthesized terpenoid derivates displayed in vitro anti-T. cruzi activity and nontoxic effects upon host cells. In infected mice, these derivatives reduced parasitic load and anti-T. cruzi antibodies during chronic stage.	[^Citation55]
Polyamine surface transporters and metabolism Trypanothione metabolism inhibitors	Thiazolidines	LPSF SF29 displayed trypomastigote lysis and amastigote death, probably by interfering with polyamine biosynthesis and consequently trypanothione biosynthesis, leading to increased sensitivity to oxidative metabolism.	[^Citation96]
	Tetradentated polyamine complexes	Tetraamines presented anti-T. cruzi activity in vitro and in vivo with low toxicity.	[^Citation97]

Abbreviations: BNZ, benznidazole; NO, nitric oxide; ROS, reactive oxygen species.

Figure 2 Chemical structures of the most promising azole derivatives that induced cure with parasite elimination in mice.

Figure 3 Chemical structures of the miscellaneous classes of most promising compounds with different mechanisms of action that induced cure with parasite elimination in mice.

Figure 4 Schematic representation of a trypomastigote of Trypanosome cruzi and the main cellular targets of the investigational compounds in the pre-clinical phase of development.

Notes: The selected targets are in blue boxes and compounds are in rose boxes. Organelles: Kp: kinetoplast, f: flagellum, fp: flagellar pocket, mt: mitochondrion, nu: nucleus, ER: endoplasmic reticulum, Go: golgi apparatus, Ac: acidocalcisomes, Gl: glycosomes, Ap: autophagosomes. The drawing is based on data from Benaim et al and Vannier-Santos et al.^163,228

Table 2 Promising Natural Products in Experimental Treatment of Trypanosoma cruzi Infections

Plant	Natural Product	Outcome	References
Aristeguietia glutinosa	Secondary metabolites isolated from the hydro-ethanolic extract of the aerial parts	The substances displayed low in vitro toxicity and anti- T. cruzi effect in infected mice by inhibiting parasite mitochondrial dehydrogenases and biosynthesis of membrane sterols.	[^Citation128]
Arrabidaea brachypoda	Dimeric flavonoids from roots	Flavonoids were active against T. cruzi in vitro and in infected mice.	[^Citation129]
Carica papaya	Crude seed extracts	Extracts were able to reduce parasitemia, but not the cardiac amastigotes nests of infected mice.	[^Citation127]
Delphinium staphisagria	Flavonoids from aerial parts	Flavonoids were active against T. cruzi in vitro and in infected mice.	[^Citation124]
Artemisia annua	artemisinin (sesquiterpene lactone)	Artemisinin presented in vitro activity and inhibited calcium-dependent ATPase activity in T. cruzi membranes.	[^Citation136]
Lychnophora passerina	goyazensolide (Sesquiterpene lactone from the aerial parts)	Goyazensolide was active in vitro and led to parasitemia reduction and negativation in parasitological tests in mice infection by Y and CL strains.	[^Citation145]
Lychnophora trichocarpha	Lychnopholide (LYC) and LYC-loaded nanocapsules (sesquiterpene lactone)	Nanocapsules containing lychnopholide reduced parasitemia and mortality in murine infection and induced cure in mice infected with T. cruzi strains resistant and sensitive to BNZ in acute and chronic phases of infection. LYC-nanocapsules formulation prevented cardiotoxicity in long term treatment.	[^Citation138^–^Citation140],[^Citation144]
Mikania species	Deoxymikanolide and other sesquiterpene lactones	Deoxymikanolide showed the highest selectivity index and induced parasitemia and mortality reduction in infected mice.	[^Citation141]
Tithonia diversifolia	Tagitinin C a sesquiterpene lactone isolated from leaves	Tagitinin C presented in vitro and in vivo activity alone and combined with BNZ.	[^Citation143]
Physalis angulata	Physalins (secosteroids) and concentrated ethanolic extract	Physalins reduced the invasion process in vitro, and intracellular parasite load, alone or in combination with BNZ. Concentrated ethanolic extract showed in vitro and in vivo activity alone and associated with BNZ.	[^Citation126,Citation130]
Piper jericoense	Furofuran lignan	Furofuran lignan was active in vitro and reduced parasitemia levels in infected mice. The compound affected the parasite structure without altering the energetic metabolism.	[^Citation132]
Salvia gilliesii	Isolated diterpene	Compound reduced parasite load and increased survival of infected mice.	[^Citation131]
Senna villosa	(8-hydroxymethylen)-trieicosanyl acetate	Isolated compound showed in vitro and in vivo activity against T. cruzi.	[^Citation122,Citation125]
Zanthoxylum chiloperone	Canthin-6-one alkaloids and leaves extract	Canthin-6-one alkaloids exhibited trypanocidal activity in vitro and in mouse model of acute or chronic infection. Leaves extract reduced parasitaemia in vivo.	[^Citation121,Citation123]
Zanthoxylum naranjillo	Lignans isolated from the leaves	Lignans displayed in vitro and in vivo activities.	[^Citation120]

Abbreviation: BNZ, benznidazole.

Table 3 Drugs Repositioned in Experimental Treatment of Chagas Disease

Compound	Original Use	Outcome	References
Albaconazole	Antifungal	Albaconazole suppressed the parasite proliferation, prevented the death of infected dogs, and induced cure in animals infected by Y strain, but not in those infected by Berenice-78.	[^Citation153]
Allopurinol	Antihyperuricemic	Allopurinol was effective in reducing parasitemia and/or modifying the evolution of acute and chronic murine infection. Allopurinol derivatives displayed similar in vitro activity than pattern drug and good bioavailability properties for oral absorption.	[^Citation169,Citation170] [^Citation171]
Amiodarone	Antiarrhythmic	Amiodarone showed in vitro activity against epimastigote and amastigote. In acute treatment of infected mice, it reduced parasitemia and increased survival.	[^Citation154]
Auranofin	Antirheumatic	Auranofin showed activity against T. cruzi in vitro and reduced parasitemia and mortality in infected mice.	[^Citation173]
Benidipine	Antihypertensive	Benidipine reduced parasite load and inflammatory process in cardiac and skeletal muscle of chronically infected mice by probably acting as cruzipain inhibitor.	[^Citation161,Citation162]
Clofazimine	Antibiotic	Clofazimine reduced parasite load and inflammatory process in cardiac and skeletal muscle of chronically infected mice, likely acting as cruzipain inhibitor.	[^Citation161,Citation162]
Clomipramine	Antidepressant	Clomipramine reduce parasitemia and electrocardiographic changes and preventing myocardial structural damage in murine infection. Clomipramine improved survival by reducing the parasitic tissue load and preventing progression of cardiac damage in chronically infected mice	[^Citation174] [^Citation175]
Imatinib	Anti-cancer	Imatinib was moderately active against different strains and forms of T. cruzi	[^Citation165]
Itraconazole	Antifungal	Itraconazole led to parasitemia reduction and mortality protection of infected mice. Itraconazole treatment promoted significant parasitemia reduction in infected dog.	[^Citation146,Citation152,Citation157] [^Citation167]
Ketoconazole	Antifungal	Ketoconazole showed anti-T. cruzi in vitro activity. The drug displayed in vivo activity in infected mice.	[^Citation148,Citation149] [^Citation147,Citation150]
Posaconazole	Antifungal	Posaconazole showed potent in vitro activity and in vivo trypanocidal activity, even against multiresistant T. cruzi strains.	[^Citation151], [^Citation154^–^Citation156]
Ravuconazole	Antifungal	Ravuconazole presented efficacy to control the infection in mice and dogs.	[^Citation116,Citation117]
Fosravuconazole (E1224) prodrug of ravuconazole	Antifungal	E1224 was well tolerated and effective in suppressing parasitemia with cure in Y strain-infected mice.	[^Citation118]
Tipifarnib	Anticancer	Tipifarnib showed potent in vitro activity due to CYP51 T. cruzi inhibition. Tipifarnib analog had potent suppressive activity on parasitemia in infected mice.	[^Citation159] [^Citation158,Citation160]

Table 4 Promising Drug Combinations in Experimental Treatment of Chagas Disease

Combination	Outcome	References
BNZ/VFV	Combination presented promising results on parasitic blood load and cure levels in infected mice.	[^Citation119]
BNZ/itraconazole	Combination improved parasitemia reduction and total survival, reduced heart qPCR positivity and cardiac damage in VL-10-infected dogs. Itraconazole improved plasma concentration of BNZ. Combination led to an improvement in parasitemia reduction, and cardiac damage in infected mice.	[^Citation176,Citation178] [^Citation177] [^Citation157]
BNZ/ketoconazole	Combination induced a synergic effect in mice infected with CL and Y T. cruzi strains, but no differences were observed in Colombian strain-infected animals.	[^Citation150]
BNZ/fosravuconazole	In vitro interaction was positive and in infected mice. Early treatment induced 100% of cure and beneficial effect on well-established infection.	[^Citation118]
BNZ/posaconazole	Shorter duration of the treatment with combination induced cure in acutely or chronically Tulahen-infected mice but did not improve the curative effect of posaconazole in Y strain infection. Sequential and combined treatments were beneficial in murine infection, decreasing the time or dose of the BNZ treatment. Combination showed same efficacy of each drug alone in Colombian strain-infected mice and improved efficacy in Brazil strain-infected mice. Treatment of acute murine infection with the combination was more effective in reducing parasitemia and myocardial injury, compared to monotherapies.	[^Citation155] [^Citation156] [^Citation56] [^Citation179]
BNZ/voriconazole	In vitro results indicated an additive interaction. In vivo, all treatments were well tolerated, and combination elicited no additional benefits over BNZ alone.	[^Citation180]
BNZ/arylimidamides	Trypanocidal activity was improved by combination therapies (BNZ plus DB766, DB289 or DB1965) in infected mice.	[^Citation87,Citation90]
BNZ/allopurinol	Allopurinol combined with low dose of BNZ had a positive interaction on serology and pathology of infected mice. Sequential treatment exhibited beneficial effects on acute and chronic infection in mice. Allopurinol plus NFX or BNZ showed in vitro synergic effect. The combinations increased the cure rate compared to BNZ alone in murine infection.	[^Citation181] [^Citation182] [^Citation172]
BNZ/ascorbic acid	Ascorbic acid combined with a low dose of BNZ improved its trypanocidal activity and attenuated the toxic effects of BNZ. Combination also reduced cardiac inflammation and hepatic damage.	[^Citation188]
BNZ/acetyl salicylic acid	Aspirin combined with lower dosages of BNZ showed better therapeutic effect in infected mice.	[^Citation193]
BNZ/clomipramine	Clomipramine improved BNZ activity in vitro and in vivo with no cell toxicity and fewer side effects	[^Citation184,Citation189]
BNZ/fenofibrate	Fenofibrate plus a low dose of BNZ attenuated cardiac dysfunction and promoted parasite clearance in mice sequentially infected with two strains of different genetic backgrounds.	[^Citation185]
BNZ/levamisole	Monotherapies of levamisole did not decrease parasitemia nor mortality rates. In combinations with a low dose of BNZ, it led to a slight improvement in the effectiveness of monotherapy	[^Citation192]
BNZ/NFX	Combining shorter treatments could cure mice, but the association led to the behavioral alterations of treated mice.	[^Citation155]
BNZ/simvastatin	Simvastatin improved BNZ activity in murine model of Chagas heart disease	[^Citation186]
BNZ/clofazimine or BNZ/benidipine	Reduced dose of 30mg/kg of BNZ associated with both drugs had the same efficacy as single 75mg/kg of BNZ in chronic model of Chagas disease in mice with no cure.	[^Citation164]
Itraconazole/amiodarone	The combination was benefic in infected dogs compared to non-treated control. The combinations were more effective in vitro T. cruzi-infection than monotherapy or BNZ; without affecting host cell metabolism and better preserving the integrity of infected cells.	[^Citation190] [^Citation191]
Posaconazole/amiodarone	Amiodarone has direct in vitro and in vivo activity against T. cruzi and showed synergic effect with posaconazole. In vitro association improved IC50 and lead alterations in the parasite.	[^Citation154] [^Citation183]
Ravuconazole/amlodipine	In vitro assays showed an additive effect for the combination. Amlodipine improved anti-T. cruzi activity of ravuconazole in infected mice.	[^Citation194]
NFX/dipyridamole	Dipyridamole potentiated the in vitro effect of NFX and improved efficacy of low dose of NFX in murine model.	[^Citation187]

Abbreviations: BNZ, benznidazole; NFX, nifurtimox.

Table 5 Drug Delivery and Nanomedicine-Based Strategies Evaluated Against Trypanosoma cruzi in Experimental Pre-Clinical Studies

Formulation	Drug/Active Molecule	Route	Outcome	Ref.
Classical formulations
Solid dispersion in poloxamer 407	BNZ	oral	15–60 mg/kg/day solid dispersion compared with classical 50 mg mg/kg/day. Same efficacy in infected mice with lower side effects in acute and chronic phases, and reduced hepatotoxicity in mice.	[^Citation203]
Solutions with co-solvents: PEG400/water and CMC/water	BNZ	oral	20–60 mg/kg/day solutions compared with classical BNZ doses produced the same efficacy in infected mice (Tulahuen strain)	[^Citation204]
Nanostructured lipid-based formulations
Nanostructured Lipid formulations	Amphotericin B	i.v. oral	Fungizone® deoxycholate, AmBisome® liposome, Amphocil® colloidal dispersion, Abelcet® lipid complex. AmBisome® was the most effective formulation with the lowest host toxicity, but less than BNZ and NFX. Different protocols: BALB/c mice infected T. cruzi Y/Tulahuen strains BNZ or NFX (controls) cleared blood trypomastigote more rapidly than amphotericin B (1 week × 3 weeks). AmBisome was the most effective formulation with the lowest host toxicity.	[^Citation205]
Liposome (Ambisome®)	Amphotericin B	i.p.	Efficacy in acute and chronic phases of mice infected with T. cruzi Tulahuen strain. The formulation showed efficacy in acute and chronic phases of mice infected with T. cruzi Tulahuen strain.	[^Citation206]
Liposome (Ambisome®)	Amphotericin B	i.v.	AmBisome® prolonged survival, without cure. Amastigote nests found in tissues of all mice treated, heart and brain in histopathological analysis in acute and chronic mice models (Y and CL strains). AmBisome® prolonged survival without cure of infection in repeated dose regimen.	[^Citation207]
pH-sensitive liposomes (DOPE:CHEMS)	Etanidazole	i.v.	Reduced parasitemia in infected mice with liposome formulation at lower doses versus no effect of free drug. Liposome showed in vitro activity against amastigote and reduced parasitemia of infected mice.	[^Citation209]
Nanoarchaeosomes	Imiquimod		Infected mice (RA strain) treated with the formulation in acute phase had improved survival and showed parasitemia reduction. However, efficacy was lower than BNZ classical treatment with 100mg/kg/day.	[^Citation218]
Self-emulsifying drug delivery system (SEDDS)	BNZ	oral	Toxicity and efficacy similar to free-BNZ. Practical and personalized orally administered liquid dosage form.	[^Citation212]
Self-emulsifying drug delivery system (SEDDS)	BNZ	oral	Formulation was safe for mice. No additional drug toxicity in infected mice (Y strain) in 20 days at doses of 100 mg/kg/day was observed. Oral BNZ-SEDDS increased BNZ AUC.	[^Citation210]
Self-emulsifying drug delivery system (SEDDS)	Ravuconazole	oral	SEDDS demonstrated low in vitro and in vivo toxicity and improved ravuconazole activity in vitro and reduced toxicity in vivo. No formulation and drug toxicity was observed in mice.	[^Citation211]
Polymeric-based nanomedicines
Poly-aggregated Amphotericin B in albumin microspheres	Amphotericin B	oral	T. cruzi-infected mice (Y strain) treated for 10 days (10–15 mg/kg/day) reduced 75% parasitemia and prolonged survival. Unable to cure mice.	[^Citation217]
Nanospheres of polyethylene glycol-polylactide	Bis-triazole D0870	i.v.	T. cruzi-infected mice treated for 30 days (3 mg/kg/day) had cure rate of 90% for CL strain and 60% for Y strain in the acute phase of infection	[^Citation216]
Nanocapsules of polyethylene glycol-polylactide	Lychnopholide (LYC) (sesquiterpene lactone)	i.v. oral	Formulation induced high cure levels in acute or chronic mice infection, including drug resistant strains and displayed minimal cardiotoxicity. Potent in vivo anti-T. cruzi activity. LYC-NC reduced cardiotoxicity compared with free-LYC. In vivo LYC-NC effects include: cure rates of 100% by PCR analysis in tissue; it induced cure in mice infected with Colombian strain and in mice in chronic phase of infection; nanocapsules increased AUC of LYC in 12-fold; high cure rates and reduced inflammation in heart of mice infected by Y, CL, VL10 and Colombian strains.	[^Citation138^–^Citation140],[^Citation144]
Micelles of polyoxyethylene-polyoxypropylene block copolymer (poloxamer 188)	Benznidazole	oral	Acute treatment of infected mice decreased heart inflammation and anti-T. cruzi specific antibodies levels. Intermittent treatments of mice with chronic infection were as effective as daily treatment. Nicaragua strain-infected mice (C57BL/6) treated (25–75mg/kg/day) during the chronic phase: low doses of BNZ-nanoparticles treatment (25 mg/kg/day) resulted in 40% negative PCR in the immunosuppressed mice. Continuous 30 consecutive days and intermittent regime once time week during 13 weeks for 7 days showed similar reduction of parasitemia compared to classical regime.	[^Citation197,Citation213]
Nanoparticles of poly-epsilon-caprolactone	Ursolic acid	i.v.	In vivo anti-T. cruzi activity led to reduction of parasitemia levels inT. cruzi-infected mice (C57BL/6), Y strain. Mice treated during the acute phase during 7 days with ursolic acid-nanoparticles showed similar reduction of parasitemia compared to BNZ. Reduced hepatotoxicity for nanoparticles.	[^Citation214]
Microparticles of poly(D,L-lactide-co-glycolide)	(-)-Hinokinin	s.c.	T. cruzi infected mice (clone CLB5) treated 20 days at 40mg/kg with Hinokinin-nanoparticles and 20 mg/kg/day with free-Hinokinin. Blood parasitemia was slightly reduced with Hinokinin-nanoparticles by fresh blood examination. No mice cure was reported.	[^Citation215]

Abbreviations: i.v, intravenous; i.p., intraperitoneal; s.c., subcutaneous; BNZ, benznidazole; NFX, nifurtimox.

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