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Efficacy of Lychnopholide Polymeric Nanocapsules after Oral and Intravenous Administration in Murine Experimental Chagas Disease Carlos Geraldo Campos de Mello,a Renata Tupinambá Branquinho,a,b Maykon Tavares Oliveira,b Matheus Marques Milagre,a Dênia Antunes Saúde-Guimarães,a,c Vanessa Carla Furtado Mosqueira,a,c Marta de Lanaa,b,d Programa de Pós-Graduação em Ciências Farmacêuticas, Escola de Farmácia, Universidade Federal de Ouro Preto, Minas Gerais, Brazila; Núcleo de Pesquisa em Ciências Biológicas (NUPEB), Universidade Federal de Ouro Preto, Minas Gerais, Brazilb; Departamento de Farmácia, Escola de Farmácia, Universidade Federal de Ouro Preto, Minas Gerais, Brazilc; Departamento de Análises Clínicas, Escola de Farmácia, Universidade Federal de Ouro Preto, Minas Gerais, Brazild
The etiological treatment of Chagas disease remains neglected. The compounds available show several limitations, mainly during the chronic phase. Lychnopholide encapsulated in polymeric nanocapsules (LYC-NC) was efficacious in mice infected with Trypanosoma cruzi and treated by intravenous administration during the acute phase (AP). As the oral route is preferred for treatment of chronic infections, such as Chagas disease, this study evaluated the use of oral LYC-NC in the AP and also compared it with LYC-NC administered to mice by the oral and intravenous routes during the chronic phase (CP). The therapeutic efficacy was evaluated by fresh blood examination, hemoculture, PCR, and enzyme-linked immunosorbent assay (ELISA). The cure rates in the AP and CP were 62.5% and 55.6%, respectively, upon oral administration of LYC–poly(D,L-lactide)–polyethylene glycol nanocapsules (LYC-PLA-PEG-NC) and 57.0% and 30.0%, respectively, with LYC–poly--caprolactone nanocapsules (LYC-PCL-NC). These cure rates were significantly higher than that of free LYC, which did not cure any animals. LYC-NC formulations administered orally during the AP showed cure rates similar to that of benznidazole, but only LYC-NC cured mice in the CP. Similar results were achieved with intravenous treatment during the CP. The higher cure rates obtained with LYC loaded in PLA-PEG-NC may be due to the smaller particle size of these NC and the presence of PEG, which influence tissue diffusion and the controlled release of LYC. Furthermore, PLA-PEG-NC may improve the stability of the drug in the gastrointestinal tract. This work is the first report of cure of experimental Chagas disease via oral administration during the CP. These findings represent a new and important perspective for oral treatment of Chagas disease.
C
hagas disease (CD) is an endemic anthropozoonosis caused by the protozoan hemoflagellate Trypanosoma cruzi (1), which infects humans and various species of mammals. T. cruzi is naturally transmitted by triatomine vectors present from the southern United States to Argentina and is widespread in 21 countries where Chagas disease is endemic (2). It is estimated that approximately 6 to 7 million individuals are currently infected (2). This neglected disease evolves to severe clinical forms that progressively disable the patient, causing sudden death due to cardiopathy. Intense human migration from areas where the disease is endemic to countries where it is not endemic contributes to CD expansion. Recently, several cases have been diagnosed in North America, Oceania, and some countries of Asia and Europe (3) due to vector-independent transmission mechanisms, such as congenital, blood transfusion, organ transplant, and other transmissions (4, 5). At this time, only two nitroheterocycle drugs are regularly used for the etiological treatment of CD, namely, benznidazole (BZ) (LAFEPE and Abarax/Elea) and nifurtimox (NFX) (Lampit/ Bayer). These drugs are effective during the recent acute and chronic phases of the infection. However, for the chronic phase, which affects most infected individuals, low rates of parasitological cure have been obtained. Additionally, severe side effects have been reported (6, 7). Considering these limitations and that CD is considered a serious public health problem in Latin America and is also present on other continents, where it is underdiagnosed, the search for new treatments is justified (8). Among 336 new therapeutic products approved from 2000 to 2011, only 4 were developed for the treatment of neglected diseases, and none for CD
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treatment, as recently reported (9). Consequently, new drugs or novel strategies with this objective are urgently needed. Natural products are an excellent source of active compounds. The Asteraceae plant family is the largest plant family known, to date, that has sesquiterpene lactones (SL) as chemical markers (10, 11). Some of these lactones have been tested in vitro and in vivo and showed significant anti-T. cruzi activities (12, 13). Our research group investigated the anti-T. cruzi efficacy of lychnopholide (LYC) in an experimental acute infection model in mice. LYC is a sesquiterpene lactone isolated from Lychnophora trichocarpha Spreng. (Asteraceae) for its anti-T. cruzi efficacy in vivo (14, 15). Interestingly, LYC also presented activity against tumor cell lines, in bacterial infections, and in gouty arthritis processes (15–17). When LYC was administered intravenously as a solution (free LYC), parasitemia was reduced and the survival rate was increased compared to those of the untreated group at the acute phase of infection for mice, although no cure was observed (18). In con-
Received 28 January 2016 Returned for modification 25 April 2016 Accepted 2 June 2016 Accepted manuscript posted online 20 June 2016 Citation de Mello CGC, Branquinho RT, Oliveira MT, Milagre MM, Saúde-Guimarães DA, Mosqueira VCF, Lana MD. 2016. Efficacy of lychnopholide polymeric nanocapsules after oral and intravenous administration in murine experimental Chagas disease. Antimicrob Agents Chemother 60:5215–5222. doi:10.1128/AAC.00178-16. Address correspondence to Marta de Lana, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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trast, when LYC was encapsulated in polymeric nanocapsules [LYC–poly-ε-caprolactone nanocapsules (LYC-PCL-NC) and LYC–poly(D,L-lactide)–polyethylene glycol nanocapsules (LYCPLA-PEG-NC)], a more pronounced parasitemia reduction and an increase of the survival rate were observed, as well as high percentages of cure in a murine model of infection with distinct T. cruzi strains (18). As previously reported, the LYC association with NC increased the substance efficacy and influenced LYC release in vitro (14). Furthermore, when sterically stabilized NC containing LYC were surface modified with covalently linked PEG chains (PLA-PEG-NC), there was a significant increase of the LYC therapeutic efficacy in vivo (18). In order to reduce the difficulties of administration of LYC in vivo (14, 18) due to its poor water solubility, conventional NC (PCL-NC) were used. Conventional NC are rapidly removed from the blood circulation via phagocytosis and then targeted passively to the liver and spleen, resulting in limited drug delivery to other organs (19). In addition, surface-modified PLA-PEG-NC were prepared to enable longer circulation in the bloodstream than that with conventional NC (19, 20). Recently, it was reported that LYC administered orally to rats showed suitable pharmacokinetic characteristics (21). Oral administration is the preferred route for the treatment of parasitic diseases, including CD. Oral therapy has a low cost and leads to better patient compliance, both of which are critical aspects of human treatment in developing countries with difficult or remote access to hospitals (8). However, when administered in conventional pharmaceutical dosage forms and exposed to the extreme pH range found along the gastrointestinal tract (GIT), the lactones are prone to chemical instability (22). Furthermore, the poor aqueous solubility of LYC can lead to low oral bioavailability in humans (23). Some authors reported that oral administration of drugs in polymeric NC offers advantages over other nanostructured systems, such as liposomes, lipid-based systems, or most micelles, because the polymeric NC are more stable in the GIT (24, 25). The polymeric wall of NC may confer a higher stability to encapsulated drugs and improve the drug body exposure (area under the concentration-time curve [AUC]) and its lymphatic transport by the oral route, as previously demonstrated (26, 27). Thus, an alternative pharmaceutical formulation involving polymeric NC loaded with LYC dissolved in the oily core was developed in order to reduce the contact of the active substance with the intestinal fluids to circumvent the above-mentioned set of difficulties (14, 18). The main focus of this article is to evaluate the efficacy of LYC administered orally and intravenously for the treatment of experimental T. cruzi (Y strain) infection in mice during the acute and chronic phases. We compared the efficacies of the LYC solution (free LYC) and LYC loaded into NC stabilized by two types of polymers, namely, PCL for conventional NC and PLA-PEG for sterically stabilized NC. This is the first study that reports the efficacy of a new drug candidate administered by the oral route against T. cruzi infections in the chronic phase. MATERIALS AND METHODS Materials. Lychnopholide (LYC) (Fig. 1) was extracted from Lychnophora trichocarpha, purified, and characterized as previously published (14, 15). Benznidazole (BZ) (N-benzyl-2-nitro-1-imidazole-acetamide) was obtained as benznidazole tablets from LAFEPE, Brazil. Poly-ε-caprolactone (PCL), with an average Mn of 42,500 g/mol, and Poloxamer 188 (Pluronic
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FIG 1 Chemical structure of lychnopholide. F-68) were purchased from Sigma. Monomethoxypolyethylene glycolblock-poly(D,L-lactide) (PLA-PEG), with an average Mn of 49,000 g/mol and a PEG block with an Mn of 5,000 g/mol, was obtained from Alkermes (Cambridge, MA) and used without further purification. The polymer poly(D,L-lactide) (PLA) (Resomer R203S; inherent viscosity, 0.25 to 0.35 dl/g) was provided by Boehringer Ingelheim (Germany) and has a molecular mass of approximately 18,000 to 28,000 Da. Miglyol 810N (capric/ caprylic triglyceride) was provided by Hulls (Germany). Epikuron 170 (soy lecithin with approximately 70% phosphatidylcholine) was purchased from Cargill (Le Blanc Mesnil, France). Ethyl acetate and N,Ndimethylacetamide (DMA) were provided by Tedia (Rio de Janeiro, Brazil). Analytical-grade PEG 300 and acetone were obtained from Vetec (Rio de Janeiro, Brazil). Preparation of lychnopholide nanocapsules. Lychnopholide loaded in conventional NC (LYC-PCL-NC) and in sterically stabilized NC (LYCPLA-PEG-NC) was prepared via interfacial deposition of the preformed polymer followed by solvent displacement as described previously (18). Briefly, 20 mg of LYC, 80 mg of PCL polymer, 250 l of Miglyol 810N, and 75 mg of Epikuron 170 were dissolved in 10 ml of acetone. This solution was poured into 20 ml of aqueous solution containing 75 mg of Poloxamer 188. The NC formed kinetically, and after 10 min of agitation, the solvents were removed and the volume reduced to 10 ml under reduced pressure. The PLA-PEG diblock polymer (60 mg) blended with 60 mg of PLA homopolymer was used to prepare PLA-PEG-NC, in this case in the absence of Poloxamer 188. Empty (blank) NC were prepared as controls. The mean hydrodynamic diameter and the polydispersity index of the NC population were determined in triplicate by dynamic light scattering (Zetasizer Nano ZS instrument; Malvern Instruments, United Kingdom), with dilution of samples 1:1,000 in Milli-Q water. The zeta potentials of the colloidal dispersions were determined by laser Doppler anemometry associated with microelectrophoresis, with dilution of samples 1:1,000 in 1 mM NaCl. Values are reported as means ⫾ standard deviations for at least three different batches of each NC formulation. The NC were filtered through 0.45-m sterile filters (25-mm Millex filter; Millipore) immediately after preparation to remove possible aggregates and were administered orally (by gavage) or intravenously, without further purification. Preparation of LYC and BZ intravenous solutions and oral formulations. LYC (2 mg/ml) and BZ (4 mg/ml) solutions suitable for intravenous administration were prepared as previously described (18) and filtered through sterile 0.22-m filters. Briefly, LYC or BZ was dissolved in a 4:6 (vol/vol) DMA-PEG 300 mixture and further diluted in an isotonic glucose solution to attain a final concentration suitable for intravenous administration. Control groups received the excipients of the intravenous solution. The LYC solution was also administered orally for comparison of different administration routes. BZ suspensions of crushed BZ tablets were dispersed in water containing 4% (wt/vol) methylcellulose for oral administration to mice by gavage.
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TABLE 1 Treatment schedules for mice infected with Trypanosoma cruzi Y strain and treated during the acute and chronic phases of infection Infection phase, route of drug administration
Experimental groupa
Dose (mg/kg/day)b
Acute or chronic, oral
Free LYC LYC-PCL-NC LYC-PLA-PEG-NC BZ INT DMA-PEG Blank NC
5 5 5 100 0 ⫹ ⫹
Chronic, intravenous
Free LYC LYC-PCL-NC LYC-PLA-PEG-NC BZ INT DMA-PEG Blank NC
2 2 2 50 0 ⫹ ⫹
a Free LYC, lychnopholide dissolved in DMA-PEG solution; LYC-PCL-NC, lychnopholide loaded in conventional nanocapsules; LYC-PLA-PEG-NC, lychnopholide loaded in sterically PEG-stabilized nanocapsules; BZ, benznidazole; INT, infected and not treated; DMA-PEG, control solution; blank NC, unloaded NC. b ⫹, the amount of excipients used in the DMA-PEG solution and unloaded NC was the same as that used in the BZ and LYC-NC formulations.
Parasites, animal ethics, and efficacy study. Parasites of the Y strain of T. cruzi, belonging to discreet typing unit (DTU) 2 (28) and considered partially resistant to treatment with BZ and NFX (29), were used. Female Swiss mice aged 28 to 30 days and with 20 to 25 g of body weight were used and maintained according to the guidelines established by CONCEA (National Council on Animal Experimentation Control). The experiments were approved by the Ethical Committee on Animal Experimentation of the Universidade Federal de Ouro Preto, Brazil (CEUA-UFOP; approval no. 2009/13). Acute infection model. For the experiments during the acute phase, animals were inoculated intraperitoneally with 10,000 blood trypomastigotes counted as described in the literature (30). The infection was confirmed in all animals at 4 days postinoculation by fresh blood examination (FBE) (30). Chronic infection model. For the experiments during the chronic phase of the infection, mice were inoculated intraperitoneally with the same T. cruzi strain (Y), but with 500 blood trypomastigotes (30). This procedure allows natural development of the chronic phase of infection in mice. For all animals, the infection was confirmed by FBE between 20 and 23 days postinoculation. Experimental groups and treatment schedules. The dose of LYC used in all experiments was determined previously (18). The experimental design and treatment schedules for the different groups of animals are shown in Table 1. Treatment during acute infection. The infected animals were divided into groups of 8 animals each and received LYC formulations (free LYC, LYC-PCL-NC, and LYC-PLA-PEG-NC) with LYC doses of 5 mg/kg of body weight. Treatment was initiated on the first day of patent parasitemia (4th day after inoculation) for up to 20 consecutive days. In parallel, a group of animals was treated with BZ on the classical schedule (30) for a murine model (100 mg/kg), with BZ administered daily by the oral route via gavage (0.2 ml) (Table 1). Treatment during chronic infection. In the chronic infection model, the treatment started at 90 days postinoculation and was maintained for 20 consecutive days. Groups of mice were treated by the oral route (gavage) or the intravenous route (tail vein). Three groups of 10 animals each received different oral LYC formulations (free LYC, LYC-PCL-NC, and
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LYC-PLA-PEG-NC) with LYC doses of 5 mg/kg/day. Equivalent groups of animals were treated intravenously with the same formulations of LYC at 2 mg/kg/day (Table 1). For comparison of treatments with LYC formulations and BZ, two groups of 10 mice each received daily BZ doses of 100 mg/kg/day (0.1 ml) and 50 mg/kg/day (0.2 ml), by the oral and intravenous routes, respectively. In parallel, the following control groups of infected animals were also evaluated: untreated animals (infected and not treated [INT]), animals treated with solution excipients (DMA-PEG), and animals treated with blank NC. Posttreatment evaluation. The efficacies of the formulations in treated mice were evaluated according to the following criteria. (i) Parasitemia. The level of parasitemia was determined by the FBE method (30). Starting on the 4th day postinfection, blood was collected daily from the caudal veins of mice for counting of the numbers of parasites in the peripheral blood of the animals. The level of parasitemia was checked during treatment and until 30 days posttreatment (dpt). (ii) Survival rates. Animals were monitored daily, and the survival rates of the animals treated during the acute phase were evaluated daily for up to 180 dpt, the time established as the end of the experiments because this was the time required for negative results in the examinations used as cure controls. (iii) HC. Hemoculture (HC) was carried out 30 days after the end of treatment, as described previously (29). Blood samples were collected via the orbital venous plexus and examined via optical microscopy for parasite detection 30, 60, 90, and 120 days after culture. (iv) PCR. Blood samples (0.2 ml/sample) were collected via the orbital venous plexus at 60 and 90 dpt and immediately added to 0.4 ml of a guanidine solution, composed of 6 M guanidine-HCl and 0.2 M EDTA at pH 8.0 (31), and stored at room temperature. DNA was extracted with a DNA purification kit (Wizard Genomic kit; Promega). PCR was performed as described previously (32), with some modifications, using the primers S35 (5=-AAATAATGTACGGGKGAGATGCATGA-3=) and S36 (5=-GGTTCGA TTGGGGTTGGTGTAATATA-3=) (Invitrogen, São Paulo, Brazil) in the presence of Platinum Taq DNA polymerase (Invitrogen, São Paulo, Brazil) to amplify kinetoplast DNA (kDNA) minicircles. Positive and negative blood sample controls were examined in parallel, as well as a PCR reagent control. All samples were processed in triplicate. (v) Enzyme-linked immunosorbent assay (ELISA). Serum samples were collected via the orbital venous plexus at 90 and 180 dpt for detection of anti-T. cruzi IgG and were analyzed as described previously (33) and modified (34). A concentration of 4.5 g/ml of antigen, a serum dilution of 1:80, and peroxidase-conjugated anti-mouse IgG at a dilution of 1:2,000 were used. The cutoff was calculated by considering the mean absorbance for 10 negative controls plus 2 standard deviations. Cure criterion. Animals were considered cured when they were simultaneously negative for all parasitological (HC and PCR) and serological (ELISA) tests, according to the classic cure criteria (6). Statistical analyses. Statistical analyses of data were performed using GraphPad Prism software v.5.0.2 (GraphPad, San Diego, CA). The Kolmogorov-Smirnov test was first performed to verify the data normality. One-way analysis of variance (ANOVA) with the Tukey posttest was used to compare areas under the curves for parasitemia versus time and survival percentages between groups. The Mann-Whitney test was used to compare maximum peak values for parasitemia. Differences between groups were considered significant if the P value was ⱕ0.05 (95% confidence interval).
RESULTS
LYC was encapsulated in NC at a high drug load, i.e., 95.4% in PCL-NC and 100% in PLA-PEG-NC. This represents an advantage because no purification step was necessary to remove unloaded LYC. The mean hydrodynamic diameters of LYC-PCL-NC and LYC-PLA-PEG-NC were 190.2 ⫾ 5.7 nm and 106.1 ⫾ 6.3 nm, respectively. These formulations were monodisperse, with a polydispersity index of ⬍0.3. Zeta potential values were ⫺32.7 ⫾ 7 mV
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FIG 2 Parasitemia curves (A) and survival curves (B) for mice infected with Trypanosoma cruzi Y strain and treated by the oral route. Free LYC, LYC-PCLNC, and LYC-PLA-PEG-NC were given at 5 mg/kg/day; BZ was given at 100 mg/kg/day. Control groups were the INT (infected and not treated), DMAPEG, and blank NC groups. All animals were treated daily for 20 consecutive days (treatment started in the patent period on the 4th day after infection).
and ⫺42.7 ⫾ 4 mV for LYC-PLA-PEG-NC and LYC-PCL-NC, respectively. No signs of general toxicity upon intravenous injection and oral gavage were observed in the behavior and physical aspects of the animals (19) receiving formulations containing LYC (free LYC, LYC-PCL-NC, and LYC-PLA-PEG-NC) or the animals in the control groups (INT, DMA-PEG, and blank NC). Animals treated during the acute phase (oral route). In this work, the Y strain, which is partially resistant to BZ, was used because it induces high parasitemia levels and mortality in mice,
which are important parameters to be evaluated in parallel to therapeutic activity. Three of eight (37.5%) animals treated with free LYC presented significant reductions (P ⬍ 0.05) in patent parasitemia compared to that in the control groups (INT, DMA-PEG, and blank NC). However, this parasitemia was higher than that observed in animals treated with LYC-PCL-NC, LYC-PLA-PEGNC, and BZ (Fig. 2A). Animals treated with LYC-PCL-NC showed significantly lower parasitemia (P ⬍ 0.05) than that observed in the control groups (Fig. 2A). All animals treated with LYC-PLA-PEG-NC (5 mg/kg/ day) and BZ (100 mg/kg/day) showed total suppression of parasitemia, which became subpatent during and after treatment (Fig. 2A and Table 1). No cure was observed in animals treated with free LYC. Animals treated with LYC-PCL-NC (5 mg/kg/day) showed a 57% (4/7 animals) cure rate, and mice treated with the LYC-PLA-PEG-NC or BZ (100 mg/kg/day) formulation displayed a cure rate of 62.5% (5/8 animals). No cure was observed in the control groups (Table 2). For the groups treated with free LYC and LYC-PCL-NC, the survival rates were 75.0% (6/8 animals) and 87.5% (7/8 animals), respectively, until 6 months posttreatment, when the mice were necropsied. For the groups treated with LYC-PLA-PEG-NC and BZ, 100% survival was recorded in the same period. No significant differences (P ⬎ 0.05) in survival rates were observed between groups of animals treated with free LYC, LYC-PCL-NC, LYCPLA-PEG-NC, and BZ formulations (Fig. 2B and Table 2). The INT control group and all other control groups showed a mean survival time of 20 days (data not shown). Animals treated during the chronic phase (oral route). The group of animals treated with free LYC showed 37.5% (3/8 animals) negative HC results, compared to 90.0% (9/10 animals) for the group treated with LYC-PCL-NC, 100.0% (10/10 animals) for the group treated with LYC-PLA-PEG-NC, and 12.5% (1/8 animals) for the group treated with BZ (100 mg/kg/day). All animals
TABLE 2 Efficacy of oral treatment with lychnopholide formulations and benznidazole in mice infected with Trypanosoma cruzi Y strain and treated during the acute and chronic phases of infection Infection phase, experimental group
Dose (mg/kg/day)d
Survival for up to 6 mo (% [no. of survivors/ no. of infected animals])
Negative laboratory test result (% [no. of negative results/no. of infected animals])
Cure (% [no. of animals cured/no. of infected animals])
Acute Free LYC LYC-PCL-NC LYC-PLA-PEG-NC BZ INT DMA-PEG Blank NC
5 5 5 100 0 ⫹ ⫹
75 (6/8) 87.5 (7/8) 100 (8/8) 100 (8/8) 0 (0/8) 0 (0/8) 0 (0/8)
FBEa 37.5 (3/8) 100 (8/8) 100 (8/8) 100 (8/8) 0 (0/8) 0 (0/8) 0 (0/8)
HC/PCRb 66.7 (4/6) 25 (2/8) 25 (2/8) 25 (2/8)
ELISAc 0 (0/6) 57 (4/7) 62.5 (5/8) 62.5 (5/8)
0 (0/6) 57 (4/7) 62.5 (5/8) 62.5 (5/8) 0 (0/8) 0 (0/8) 0 (0/8)
Chronic Free LYC LYC-PCL NC LYC-PLA-PEG-NC BZ INT DMA-PEG Blank NC
5 5 5 100 0 ⫹ ⫹
70 (7/10) 100 (10/10) 90 (9/10) 80 (8/10) 70 (7/10) 70 (7/10) 70 (7/10)
HCb 37.5 (3/8) 90 (9/10) 100 (10/10) 12.5 (1/8) 0 (7/7) 0 (7/7) 0 (7/7)
PCRb 28.6 (2/7) 70 (7/10) 88.9 (8/9) 12.5 (1/8)
ELISAc 0 (0/7) 30 (3/10) 55.6 (5/9) 0 (0/8) 0 (0/7) 0 (0/7) 0 (0/7)
0 (0/7) 30 (3/10) 55.6 (5/9) 0 (0/8) 0 (0/7) 0 (0/7) 0 (0/7)
a
FBE was performed during treatment and at 30 dpt. HC and PCR were performed at 30 dpt and at 60 and 90 dpt, respectively. c ELISA was performed at 90 and 180 dpt. d ⫹, the amount of excipients used for the DMA-PEG solution and blank NC was the same as that used in the BZ and LYC-NC formulations. b
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TABLE 3 Efficacy of intravenous treatment with LYC formulations and BZ in mice with chronic infection with Trypanosoma cruzi Y strain
Experimental group
Dose (mg/kg/day)d
Survival for up to 6 mo (% [no. of survivors/ no. of infected animals])
Free LYC LYC-PCL-NC LYC-PLA-PEG-NC BZ INT DMA-PEG Blank NC
2 2 2 50 0 ⫹ ⫹
40 (4/10) 90 (9/10) 100 (10/10) 50 (5/10) 50 (5/10) 60 (6/10) 50 (5/10)
Negative laboratory test result (% [no. of negative results/no. of infected animals]) HCa
PCRb
ELISAc
Cure (% [no. of animals cured/no. of infected animals])
50 (3/6) 100 (9/9) 80 (8/10) 100 (6/6) 0 (0/5) 0 (0/6) 0 (0/5)
17.7 (1/6) 77.8 (7/9) 60 (6/10) 66.7 (4/6)
0 (0/4) 33.3 (3/9) 50 (5/10) 0 (0/5) 0 (0/5) 0 (0/6) 0 (0/5)
0 (0/4) 33.3 (3/9) 50 (5/10) 0 (0/5) 0 (0/5) 0 (0/6) 0 (0/5)
a
HC was performed at 30 dpt. PCR was performed at 60 and 90 dpt. c ELISA was performed at 90 and 180 dpt. d ⫹, the amount of excipients used for the DMA-PEG solution and blank NC was the same as that used in the BZ and LYC-NC formulations. b
(100%) of the control groups were positive by HC (Table 2). PCR was negative for 28.6% (2/7 animals) of the animals treated with free LYC, 70.0% (7/10 animals) of those treated with LYC-PCLNC, 88.9% (8/9 animals) of those treated with LYC-PLA-PEGNC, and 12.5% (1/8 animals) of those treated with BZ (Table 2). All animals treated with free LYC or BZ and all animals in the control groups (INT, DMA-PEG, and blank NC) were ELISA positive for anti-T. cruzi IgG. ELISA was negative for 30% (3/10 animals) of the animals treated with LYC-PCL-NC and 55.6% (5/9 animals) of those treated with LYC-PLA-PEG-NC (Table 2). Considering the cure criterion adopted, no cure was observed in animals treated with free LYC or BZ or in the control groups (Table 2). Animals treated with LYC-PCL-NC and LYC-PLAPEG-NC showed cure rates of 30% (3/10 animals) and 55.6% (5/9 animals), respectively (Table 2). Animal survival rates for up to 6 months posttreatment are illustrated in Table 2. The percentages of survival for the groups of mice treated with different formulations were as follows: free LYC, 70% (7/10 animals); LYC-PCL-NC, 100% (10/10 animals); LYCPLA-PEG-NC, 90% (9/10 animals); and BZ, 80% (8/10 animals) (Table 2). The untreated control groups (INT, DMA-PEG, and blank NC) all showed a survival rate of 70% (7/10 animals) (Table 2). Animals treated during the chronic phase (intravenous route). For the group of animals treated with free LYC (2 mg/kg/ day), 50% (3/6 animals) were negative by HC, compared to 100% (9/9 animals) of the animals treated with LYC-PCL-NC, 80% (8/10 animals) of those treated with LYC-PLA-PEG-NC, and 100% (6/6 animals) of those treated with BZ. All animals of the control groups were positive by HC (Table 3). PCR was negative for 17.7% (1/6 animals) of the animals treated with free LYC, 77.8% (7/9 animals) of those treated with LYC-PCL-NC, 60% (6/10 animals) of those treated with LYCPLA-PEG-NC, and 66.7% (4/6 animals) of those treated with BZ (Table 3). All animals treated with free LYC or BZ (50 mg/kg/day) or in the control groups were ELISA positive. ELISA was negative for 33.3% (3/9 animals) of the animals treated with LYC-PCL-NC and 50% (5/10 animals) of the animals treated with LYC-PLAPEG-NC (Table 3). No cure was observed in the animals of the free LYC, BZ, and control groups (Table 3). Considering the cure criterion adopted, animals treated with LYC-PCL-NC and LYC-PLA-PEG-NC showed cure rates of 33.3% (3/9 animals) and 50% (5/10 animals), respectively (Table 3).
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Animal survival rates for up to 6 months posttreatment are illustrated in Table 3. The survival rates for animals treated with the different formulations were as follows: free LYC, 40% (4/10 animals); LYC-PCL-NC, 90% (9/10 animals); LYC-PLA-PEGNC, 100% (10/10 animals); and BZ, 50% (5/10 animals). The untreated control (INT) group and the blank NC group both showed a survival rate of 50% (5/10 animals), similar to that observed for the DMA-PEG group (60% [6/10 animals]). DISCUSSION
Despite the large number of new tested compounds that have shown anti-T. cruzi activity in vitro and efficacy in vivo, only four molecules have been subjected to clinical trials for CD. In general, the results with these drugs were contradictory or even unsatisfactory (7, 35, 36). In an attempt to overcome the limitations of CD therapy, some researchers recently suggested the development of drug delivery systems using nanocarriers to improve the drug pharmacological effects (35, 37) by controlling drug release, reducing toxicity, and modulating the pharmacokinetics and biodistribution (8, 35, 37, 38). Few studies have investigated the association of drugs or chemical leads with polymeric nanocarriers for the treatment of CD. D0870, a bis-triazole compound, was able to improve the cure rate to 60% for animals infected with the Y strain (38). Other examples are the association of BZ with self-emulsifying delivery systems (SNEDDS), which increased the index of cure for Swiss mice infected with the same T. cruzi strain (35). Furthermore, the natural isolated compound lychnopholide (LYC) associated with polymeric NC was highly efficacious against T. cruzi infection in mice (18). A recent study with rats suggested the favorable use of oral LYC as an active substance or pharmacological agent because of its physicochemical properties of good permeability and rapid absorption, distribution, and elimination, resulting in 68% oral bioavailability (21). However, LYC has poor solubility, and its fast elimination implies its possible administration in repeated doses. Controlled-release formulations, such as NC, may improve the pharmacokinetics and biodistribution and thus maintain efficacy with fewer doses. For our studies, we selected the murine model because it is considered the most susceptible animal model for experimental T. cruzi infection and is the most widely used model for screening of drugs for CD (29, 39). A recent experience of our group in the intravenous treatment of Swiss mice infected with the Y strain of T. cruzi with 2 mg/kg/
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day of LYC in NC for 20 consecutive days resulted in significantly better efficacy than that with free LYC or BZ (18). Considering these previous results and the importance of the oral route of administration for potential compounds against chronic CD, we decided to compare in the present study the oral administration of LYC-NC formulations for the treatment of the acute and chronic phases of infection in mice infected with the T. cruzi Y strain with the results obtained for the chronic phase upon intravenous administration of the drug. In order to avoid toxicity, the dose of 5 mg/kg was chosen for oral administration. The NC formulations (LYC-PLA-PEG-NC and LYC-PCLNC) presented a bluish milky aspect characteristic of colloidal suspensions. The experimental average particle diameters of the formulations, ranging from 106 to 190 nm, and a low polydispersity index (40) warrant safety by intravenous administration and a low risk of embolic events. LYC-PLA-PEG-NC showed reduced sizes compared to those of LYC-PCL-NC (18). The smaller size of LYC-PLA-PEG-NC could be attributed to a difference in the arrangement of the hydrophilic block of the PEG polymer on the NC surface, which influences interfacial tension between solvents during their mixing, generating particles of smaller size. Moreover, NC of small size are more prone to have access to infectious and inflammatory foci due to their ability to diffuse through the leaky endothelium (41). This aspect may be particularly interesting for CD treatment. The oral administration of LYC at 5 mg/kg/day loaded in PLAPEG-NC during the acute phase in mice infected with T. cruzi strain Y led to a higher efficacy than those for all other groups. LYC-PCL-NC and BZ also led to significant parasitemia reductions compared to those for the free LYC and control groups. The survival of infected animals treated with LYC-PLA-PEG-NC and BZ was total and higher than those for the groups treated with LYC-PCL-NC and free LYC, but survival was null for the control groups. In addition, the cure rates observed were higher with LYC-PLA-PEG-NC (62.5%) and BZ (62.5%) and similar to that with LYC-PCL-NC (57%). However, our previous results showed higher cure rates (18) when animals infected with the same T. cruzi strain were treated via intravenous administration with the same LYC-NC formulations during the acute phase of the infection. A higher percentage of cure upon oral administration during the chronic phase of Y strain infection was obtained with LYCPLA-PEG-NC (55.6%) than with LYC-PCL-NC (30%), while animals in all other groups (free LYC, BZ, and controls) were not cured. Intravenous administration of the same set of formulations at a different dose (2 mg/kg/day) and under the same conditions of treatment during the chronic phase of the infection revealed cure rates of 50.0% with LYC-PLA-PEG-NC and 33.3% with LYCPCL-NC. No cure was observed for animals of the other groups (free LYC, BZ, and controls). Therefore, sterically stabilized NC, such as LYC-PLA-PEG-NC, were found to improve the cure rates of LYC, in agreement with the previously reported improvement in efficacy observed for T. cruzi-infected mice in the acute phase (18). Thus, for the chronic phase, similar indexes of cure were reached between intravenous (50% and 33.3%) and oral (55.6% and 30%) treatments for LYC-PLA-PEG-NC and LYC-PCL-NC, respectively. All experiments in the present work and previous reports by our team (18) revealed that the administration of LYC loaded in NC formulations provided higher cure rates than those for treatment with LYC in solution, regardless of the route of administration, dose, and period of the infection when treatment
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was initiated. It was again demonstrated that free LYC reduced parasitemia and increased the survival rate of mice compared to those for control groups, although none of the infected and treated animals were healed, which shows the therapeutic advantage and effectiveness of encapsulation of LYC in polymeric NC. The survival rates observed for the control groups in the chronicphase model (INT, blank NC, and DMA-PEG solution) were expected, because these animals were infected with an inoculum 20 times lower than that for mice infected for treatment of the acute phase, a condition necessary to enable chronic infection using the T. cruzi Y strain. The kinetic release profile of LYC from NC was already determined (14). The time required to release 50% (T50) of LYC from LYC-PCL-NC was found to be 20.7 h, versus a dissolution T50 of 7.3 h for LYC powder in release medium. Thus, the improved efficacy observed with LYC-PCL-NC compared to free LYC may be due to the slow release of LYC from NC, i.e., a form of sustained release. In general, the PEG corona surrounding NC reduces the rate of release of lipophilic drug to the external medium in the presence of proteins present there, as previously reported (42, 43). Furthermore, PEG prevents the access of proteins to the NC oily core, which may reduce LYC release or NC wall degradation. The significantly higher cure rates obtained with the PEG-containing formulation (LYC-PLA-PEG-NC) than with the conventional formulation (LYC-PCL-NC) by the oral route can thus be interpreted as a consequence of different rates of release and/or the additional protection against the GIT medium and the action of enzymes provided by PEG at the surface (44, 45). The differences between the cure results we observed with the sterically stabilized and conventional NC formulations are in agreement with findings reported in the literature for other drugnanocarrier systems (18, 44, 46, 47). Nanoparticles of PEG have the ability to extend the circulation time due to reduction of opsonization and avoidance of subsequent clearance by the mononuclear phagocytic system (MPS). Consequently, nanoparticles are not eliminated or metabolized as fast as free LYC and may accumulate and act in inflamed tissues, as occurs in T. cruzi infection. This may be the main cause of the improved efficacy of LYC loaded in NC. Although the oral route is the route most used for drug administration, some drawbacks are found for active chemical entities, such as LYC, that suffer degradation at extreme pH. The higher cure rate for animals infected with the Y strain of T. cruzi observed here upon oral administration of encapsulated LYC is an indication that the effect of the molecule was preserved, which in turn is an indication that the molecular integrity was maintained. Polymeric nanocarriers have the ability to interact with infected target cells via a variety of endocytic mechanisms (35) and to deliver anti-T. cruzi drugs or substances inside the cell (35, 37, 38). In the acute phase of infection, the therapeutic strategy consists of maintaining the nanoparticles in the blood due to the large number of trypomastigotes in this compartment. This can be achieved with the use of either type of NC studied here, as they both circulate longer in the blood compartment than free LYC does. On the other hand, during the chronic phase of infection, the strategy is to reach the amastigote forms of the parasite inside the cell by using small, long-circulating nanocarriers (NC) and also to reach the interstitial matrix, where the amastigotes or trypomastigotes derived from them are released. This approach takes
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Oral Lychnopholide Nanocapsules Cure Chagas Disease
advantage of the endothelial leakage of inflamed tissues, which results in nanoparticle perfusion to the extravascular region (35). In conclusion, this work is the first report of cure of experimental Chagas disease via oral administration of an isolated natural substance loaded in sterically stabilized and conventional polymeric nanocapsules in both phases of infection with a T. cruzi strain partially resistant to benznidazole. Such levels of cure have never been achieved for chronic experimental Chagas disease with a T. cruzi strain partially resistant to treatment. These findings represent a new and important perspective for treatment of Chagas disease by the oral route.
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ACKNOWLEDGMENTS We thank G. Pound-Lana for English grammar revisions. We thank also the UFOP and CAPES undergraduate and postgraduate scholarships. We have no conflicts of interest to declare.
FUNDING INFORMATION
18.
This work, including the efforts of Dênia Antunes Saúde-Guimarães, Vanessa Carla Furtado Mosqueira, and Marta de Lana, was funded by FAPEMIG as NANOBIOMG (00007-14 and 40/11), TOXIFAR (0043213), and BIOTERISMO (APQ 00432-12) networks and projects (APQ00566-11, APQ-00956-13) and partially funded by CAPES-COFECUB 768/13. Marta de Lana and Vanessa Carla Furtado Mosqueira are research fellows of Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/MCTI).
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Efficacy of Lychnopholide Polymeric Nanocapsules after Oral and Intravenous Administration in Murine Experimental Chagas Disease Carlos Geraldo Campos de Mello,a Renata Tupinambá Branquinho,a,b Maykon Tavares Oliveira,b Matheus Marques Milagre,a Dênia Antunes Saúde-Guimarães,a,c Vanessa Carla Furtado Mosqueira,a,c Marta de Lanaa,b,d Programa de Pós-Graduação em Ciências Farmacêuticas, Escola de Farmácia, Universidade Federal de Ouro Preto, Minas Gerais, Brazila; Núcleo de Pesquisa em Ciências Biológicas (NUPEB), Universidade Federal de Ouro Preto, Minas Gerais, Brazilb; Departamento de Farmácia, Escola de Farmácia, Universidade Federal de Ouro Preto, Minas Gerais, Brazilc; Departamento de Análises Clínicas, Escola de Farmácia, Universidade Federal de Ouro Preto, Minas Gerais, Brazild
The etiological treatment of Chagas disease remains neglected. The compounds available show several limitations, mainly during the chronic phase. Lychnopholide encapsulated in polymeric nanocapsules (LYC-NC) was efficacious in mice infected with Trypanosoma cruzi and treated by intravenous administration during the acute phase (AP). As the oral route is preferred for treatment of chronic infections, such as Chagas disease, this study evaluated the use of oral LYC-NC in the AP and also compared it with LYC-NC administered to mice by the oral and intravenous routes during the chronic phase (CP). The therapeutic efficacy was evaluated by fresh blood examination, hemoculture, PCR, and enzyme-linked immunosorbent assay (ELISA). The cure rates in the AP and CP were 62.5% and 55.6%, respectively, upon oral administration of LYC–poly(D,L-lactide)–polyethylene glycol nanocapsules (LYC-PLA-PEG-NC) and 57.0% and 30.0%, respectively, with LYC–poly--caprolactone nanocapsules (LYC-PCL-NC). These cure rates were significantly higher than that of free LYC, which did not cure any animals. LYC-NC formulations administered orally during the AP showed cure rates similar to that of benznidazole, but only LYC-NC cured mice in the CP. Similar results were achieved with intravenous treatment during the CP. The higher cure rates obtained with LYC loaded in PLA-PEG-NC may be due to the smaller particle size of these NC and the presence of PEG, which influence tissue diffusion and the controlled release of LYC. Furthermore, PLA-PEG-NC may improve the stability of the drug in the gastrointestinal tract. This work is the first report of cure of experimental Chagas disease via oral administration during the CP. These findings represent a new and important perspective for oral treatment of Chagas disease.
C
hagas disease (CD) is an endemic anthropozoonosis caused by the protozoan hemoflagellate Trypanosoma cruzi (1), which infects humans and various species of mammals. T. cruzi is naturally transmitted by triatomine vectors present from the southern United States to Argentina and is widespread in 21 countries where Chagas disease is endemic (2). It is estimated that approximately 6 to 7 million individuals are currently infected (2). This neglected disease evolves to severe clinical forms that progressively disable the patient, causing sudden death due to cardiopathy. Intense human migration from areas where the disease is endemic to countries where it is not endemic contributes to CD expansion. Recently, several cases have been diagnosed in North America, Oceania, and some countries of Asia and Europe (3) due to vector-independent transmission mechanisms, such as congenital, blood transfusion, organ transplant, and other transmissions (4, 5). At this time, only two nitroheterocycle drugs are regularly used for the etiological treatment of CD, namely, benznidazole (BZ) (LAFEPE and Abarax/Elea) and nifurtimox (NFX) (Lampit/ Bayer). These drugs are effective during the recent acute and chronic phases of the infection. However, for the chronic phase, which affects most infected individuals, low rates of parasitological cure have been obtained. Additionally, severe side effects have been reported (6, 7). Considering these limitations and that CD is considered a serious public health problem in Latin America and is also present on other continents, where it is underdiagnosed, the search for new treatments is justified (8). Among 336 new therapeutic products approved from 2000 to 2011, only 4 were developed for the treatment of neglected diseases, and none for CD
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treatment, as recently reported (9). Consequently, new drugs or novel strategies with this objective are urgently needed. Natural products are an excellent source of active compounds. The Asteraceae plant family is the largest plant family known, to date, that has sesquiterpene lactones (SL) as chemical markers (10, 11). Some of these lactones have been tested in vitro and in vivo and showed significant anti-T. cruzi activities (12, 13). Our research group investigated the anti-T. cruzi efficacy of lychnopholide (LYC) in an experimental acute infection model in mice. LYC is a sesquiterpene lactone isolated from Lychnophora trichocarpha Spreng. (Asteraceae) for its anti-T. cruzi efficacy in vivo (14, 15). Interestingly, LYC also presented activity against tumor cell lines, in bacterial infections, and in gouty arthritis processes (15–17). When LYC was administered intravenously as a solution (free LYC), parasitemia was reduced and the survival rate was increased compared to those of the untreated group at the acute phase of infection for mice, although no cure was observed (18). In con-
Received 28 January 2016 Returned for modification 25 April 2016 Accepted 2 June 2016 Accepted manuscript posted online 20 June 2016 Citation de Mello CGC, Branquinho RT, Oliveira MT, Milagre MM, Saúde-Guimarães DA, Mosqueira VCF, Lana MD. 2016. Efficacy of lychnopholide polymeric nanocapsules after oral and intravenous administration in murine experimental Chagas disease. Antimicrob Agents Chemother 60:5215–5222. doi:10.1128/AAC.00178-16. Address correspondence to Marta de Lana, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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trast, when LYC was encapsulated in polymeric nanocapsules [LYC–poly-ε-caprolactone nanocapsules (LYC-PCL-NC) and LYC–poly(D,L-lactide)–polyethylene glycol nanocapsules (LYCPLA-PEG-NC)], a more pronounced parasitemia reduction and an increase of the survival rate were observed, as well as high percentages of cure in a murine model of infection with distinct T. cruzi strains (18). As previously reported, the LYC association with NC increased the substance efficacy and influenced LYC release in vitro (14). Furthermore, when sterically stabilized NC containing LYC were surface modified with covalently linked PEG chains (PLA-PEG-NC), there was a significant increase of the LYC therapeutic efficacy in vivo (18). In order to reduce the difficulties of administration of LYC in vivo (14, 18) due to its poor water solubility, conventional NC (PCL-NC) were used. Conventional NC are rapidly removed from the blood circulation via phagocytosis and then targeted passively to the liver and spleen, resulting in limited drug delivery to other organs (19). In addition, surface-modified PLA-PEG-NC were prepared to enable longer circulation in the bloodstream than that with conventional NC (19, 20). Recently, it was reported that LYC administered orally to rats showed suitable pharmacokinetic characteristics (21). Oral administration is the preferred route for the treatment of parasitic diseases, including CD. Oral therapy has a low cost and leads to better patient compliance, both of which are critical aspects of human treatment in developing countries with difficult or remote access to hospitals (8). However, when administered in conventional pharmaceutical dosage forms and exposed to the extreme pH range found along the gastrointestinal tract (GIT), the lactones are prone to chemical instability (22). Furthermore, the poor aqueous solubility of LYC can lead to low oral bioavailability in humans (23). Some authors reported that oral administration of drugs in polymeric NC offers advantages over other nanostructured systems, such as liposomes, lipid-based systems, or most micelles, because the polymeric NC are more stable in the GIT (24, 25). The polymeric wall of NC may confer a higher stability to encapsulated drugs and improve the drug body exposure (area under the concentration-time curve [AUC]) and its lymphatic transport by the oral route, as previously demonstrated (26, 27). Thus, an alternative pharmaceutical formulation involving polymeric NC loaded with LYC dissolved in the oily core was developed in order to reduce the contact of the active substance with the intestinal fluids to circumvent the above-mentioned set of difficulties (14, 18). The main focus of this article is to evaluate the efficacy of LYC administered orally and intravenously for the treatment of experimental T. cruzi (Y strain) infection in mice during the acute and chronic phases. We compared the efficacies of the LYC solution (free LYC) and LYC loaded into NC stabilized by two types of polymers, namely, PCL for conventional NC and PLA-PEG for sterically stabilized NC. This is the first study that reports the efficacy of a new drug candidate administered by the oral route against T. cruzi infections in the chronic phase. MATERIALS AND METHODS Materials. Lychnopholide (LYC) (Fig. 1) was extracted from Lychnophora trichocarpha, purified, and characterized as previously published (14, 15). Benznidazole (BZ) (N-benzyl-2-nitro-1-imidazole-acetamide) was obtained as benznidazole tablets from LAFEPE, Brazil. Poly-ε-caprolactone (PCL), with an average Mn of 42,500 g/mol, and Poloxamer 188 (Pluronic
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FIG 1 Chemical structure of lychnopholide. F-68) were purchased from Sigma. Monomethoxypolyethylene glycolblock-poly(D,L-lactide) (PLA-PEG), with an average Mn of 49,000 g/mol and a PEG block with an Mn of 5,000 g/mol, was obtained from Alkermes (Cambridge, MA) and used without further purification. The polymer poly(D,L-lactide) (PLA) (Resomer R203S; inherent viscosity, 0.25 to 0.35 dl/g) was provided by Boehringer Ingelheim (Germany) and has a molecular mass of approximately 18,000 to 28,000 Da. Miglyol 810N (capric/ caprylic triglyceride) was provided by Hulls (Germany). Epikuron 170 (soy lecithin with approximately 70% phosphatidylcholine) was purchased from Cargill (Le Blanc Mesnil, France). Ethyl acetate and N,Ndimethylacetamide (DMA) were provided by Tedia (Rio de Janeiro, Brazil). Analytical-grade PEG 300 and acetone were obtained from Vetec (Rio de Janeiro, Brazil). Preparation of lychnopholide nanocapsules. Lychnopholide loaded in conventional NC (LYC-PCL-NC) and in sterically stabilized NC (LYCPLA-PEG-NC) was prepared via interfacial deposition of the preformed polymer followed by solvent displacement as described previously (18). Briefly, 20 mg of LYC, 80 mg of PCL polymer, 250 l of Miglyol 810N, and 75 mg of Epikuron 170 were dissolved in 10 ml of acetone. This solution was poured into 20 ml of aqueous solution containing 75 mg of Poloxamer 188. The NC formed kinetically, and after 10 min of agitation, the solvents were removed and the volume reduced to 10 ml under reduced pressure. The PLA-PEG diblock polymer (60 mg) blended with 60 mg of PLA homopolymer was used to prepare PLA-PEG-NC, in this case in the absence of Poloxamer 188. Empty (blank) NC were prepared as controls. The mean hydrodynamic diameter and the polydispersity index of the NC population were determined in triplicate by dynamic light scattering (Zetasizer Nano ZS instrument; Malvern Instruments, United Kingdom), with dilution of samples 1:1,000 in Milli-Q water. The zeta potentials of the colloidal dispersions were determined by laser Doppler anemometry associated with microelectrophoresis, with dilution of samples 1:1,000 in 1 mM NaCl. Values are reported as means ⫾ standard deviations for at least three different batches of each NC formulation. The NC were filtered through 0.45-m sterile filters (25-mm Millex filter; Millipore) immediately after preparation to remove possible aggregates and were administered orally (by gavage) or intravenously, without further purification. Preparation of LYC and BZ intravenous solutions and oral formulations. LYC (2 mg/ml) and BZ (4 mg/ml) solutions suitable for intravenous administration were prepared as previously described (18) and filtered through sterile 0.22-m filters. Briefly, LYC or BZ was dissolved in a 4:6 (vol/vol) DMA-PEG 300 mixture and further diluted in an isotonic glucose solution to attain a final concentration suitable for intravenous administration. Control groups received the excipients of the intravenous solution. The LYC solution was also administered orally for comparison of different administration routes. BZ suspensions of crushed BZ tablets were dispersed in water containing 4% (wt/vol) methylcellulose for oral administration to mice by gavage.
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TABLE 1 Treatment schedules for mice infected with Trypanosoma cruzi Y strain and treated during the acute and chronic phases of infection Infection phase, route of drug administration
Experimental groupa
Dose (mg/kg/day)b
Acute or chronic, oral
Free LYC LYC-PCL-NC LYC-PLA-PEG-NC BZ INT DMA-PEG Blank NC
5 5 5 100 0 ⫹ ⫹
Chronic, intravenous
Free LYC LYC-PCL-NC LYC-PLA-PEG-NC BZ INT DMA-PEG Blank NC
2 2 2 50 0 ⫹ ⫹
a Free LYC, lychnopholide dissolved in DMA-PEG solution; LYC-PCL-NC, lychnopholide loaded in conventional nanocapsules; LYC-PLA-PEG-NC, lychnopholide loaded in sterically PEG-stabilized nanocapsules; BZ, benznidazole; INT, infected and not treated; DMA-PEG, control solution; blank NC, unloaded NC. b ⫹, the amount of excipients used in the DMA-PEG solution and unloaded NC was the same as that used in the BZ and LYC-NC formulations.
Parasites, animal ethics, and efficacy study. Parasites of the Y strain of T. cruzi, belonging to discreet typing unit (DTU) 2 (28) and considered partially resistant to treatment with BZ and NFX (29), were used. Female Swiss mice aged 28 to 30 days and with 20 to 25 g of body weight were used and maintained according to the guidelines established by CONCEA (National Council on Animal Experimentation Control). The experiments were approved by the Ethical Committee on Animal Experimentation of the Universidade Federal de Ouro Preto, Brazil (CEUA-UFOP; approval no. 2009/13). Acute infection model. For the experiments during the acute phase, animals were inoculated intraperitoneally with 10,000 blood trypomastigotes counted as described in the literature (30). The infection was confirmed in all animals at 4 days postinoculation by fresh blood examination (FBE) (30). Chronic infection model. For the experiments during the chronic phase of the infection, mice were inoculated intraperitoneally with the same T. cruzi strain (Y), but with 500 blood trypomastigotes (30). This procedure allows natural development of the chronic phase of infection in mice. For all animals, the infection was confirmed by FBE between 20 and 23 days postinoculation. Experimental groups and treatment schedules. The dose of LYC used in all experiments was determined previously (18). The experimental design and treatment schedules for the different groups of animals are shown in Table 1. Treatment during acute infection. The infected animals were divided into groups of 8 animals each and received LYC formulations (free LYC, LYC-PCL-NC, and LYC-PLA-PEG-NC) with LYC doses of 5 mg/kg of body weight. Treatment was initiated on the first day of patent parasitemia (4th day after inoculation) for up to 20 consecutive days. In parallel, a group of animals was treated with BZ on the classical schedule (30) for a murine model (100 mg/kg), with BZ administered daily by the oral route via gavage (0.2 ml) (Table 1). Treatment during chronic infection. In the chronic infection model, the treatment started at 90 days postinoculation and was maintained for 20 consecutive days. Groups of mice were treated by the oral route (gavage) or the intravenous route (tail vein). Three groups of 10 animals each received different oral LYC formulations (free LYC, LYC-PCL-NC, and
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LYC-PLA-PEG-NC) with LYC doses of 5 mg/kg/day. Equivalent groups of animals were treated intravenously with the same formulations of LYC at 2 mg/kg/day (Table 1). For comparison of treatments with LYC formulations and BZ, two groups of 10 mice each received daily BZ doses of 100 mg/kg/day (0.1 ml) and 50 mg/kg/day (0.2 ml), by the oral and intravenous routes, respectively. In parallel, the following control groups of infected animals were also evaluated: untreated animals (infected and not treated [INT]), animals treated with solution excipients (DMA-PEG), and animals treated with blank NC. Posttreatment evaluation. The efficacies of the formulations in treated mice were evaluated according to the following criteria. (i) Parasitemia. The level of parasitemia was determined by the FBE method (30). Starting on the 4th day postinfection, blood was collected daily from the caudal veins of mice for counting of the numbers of parasites in the peripheral blood of the animals. The level of parasitemia was checked during treatment and until 30 days posttreatment (dpt). (ii) Survival rates. Animals were monitored daily, and the survival rates of the animals treated during the acute phase were evaluated daily for up to 180 dpt, the time established as the end of the experiments because this was the time required for negative results in the examinations used as cure controls. (iii) HC. Hemoculture (HC) was carried out 30 days after the end of treatment, as described previously (29). Blood samples were collected via the orbital venous plexus and examined via optical microscopy for parasite detection 30, 60, 90, and 120 days after culture. (iv) PCR. Blood samples (0.2 ml/sample) were collected via the orbital venous plexus at 60 and 90 dpt and immediately added to 0.4 ml of a guanidine solution, composed of 6 M guanidine-HCl and 0.2 M EDTA at pH 8.0 (31), and stored at room temperature. DNA was extracted with a DNA purification kit (Wizard Genomic kit; Promega). PCR was performed as described previously (32), with some modifications, using the primers S35 (5=-AAATAATGTACGGGKGAGATGCATGA-3=) and S36 (5=-GGTTCGA TTGGGGTTGGTGTAATATA-3=) (Invitrogen, São Paulo, Brazil) in the presence of Platinum Taq DNA polymerase (Invitrogen, São Paulo, Brazil) to amplify kinetoplast DNA (kDNA) minicircles. Positive and negative blood sample controls were examined in parallel, as well as a PCR reagent control. All samples were processed in triplicate. (v) Enzyme-linked immunosorbent assay (ELISA). Serum samples were collected via the orbital venous plexus at 90 and 180 dpt for detection of anti-T. cruzi IgG and were analyzed as described previously (33) and modified (34). A concentration of 4.5 g/ml of antigen, a serum dilution of 1:80, and peroxidase-conjugated anti-mouse IgG at a dilution of 1:2,000 were used. The cutoff was calculated by considering the mean absorbance for 10 negative controls plus 2 standard deviations. Cure criterion. Animals were considered cured when they were simultaneously negative for all parasitological (HC and PCR) and serological (ELISA) tests, according to the classic cure criteria (6). Statistical analyses. Statistical analyses of data were performed using GraphPad Prism software v.5.0.2 (GraphPad, San Diego, CA). The Kolmogorov-Smirnov test was first performed to verify the data normality. One-way analysis of variance (ANOVA) with the Tukey posttest was used to compare areas under the curves for parasitemia versus time and survival percentages between groups. The Mann-Whitney test was used to compare maximum peak values for parasitemia. Differences between groups were considered significant if the P value was ⱕ0.05 (95% confidence interval).
RESULTS
LYC was encapsulated in NC at a high drug load, i.e., 95.4% in PCL-NC and 100% in PLA-PEG-NC. This represents an advantage because no purification step was necessary to remove unloaded LYC. The mean hydrodynamic diameters of LYC-PCL-NC and LYC-PLA-PEG-NC were 190.2 ⫾ 5.7 nm and 106.1 ⫾ 6.3 nm, respectively. These formulations were monodisperse, with a polydispersity index of ⬍0.3. Zeta potential values were ⫺32.7 ⫾ 7 mV
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FIG 2 Parasitemia curves (A) and survival curves (B) for mice infected with Trypanosoma cruzi Y strain and treated by the oral route. Free LYC, LYC-PCLNC, and LYC-PLA-PEG-NC were given at 5 mg/kg/day; BZ was given at 100 mg/kg/day. Control groups were the INT (infected and not treated), DMAPEG, and blank NC groups. All animals were treated daily for 20 consecutive days (treatment started in the patent period on the 4th day after infection).
and ⫺42.7 ⫾ 4 mV for LYC-PLA-PEG-NC and LYC-PCL-NC, respectively. No signs of general toxicity upon intravenous injection and oral gavage were observed in the behavior and physical aspects of the animals (19) receiving formulations containing LYC (free LYC, LYC-PCL-NC, and LYC-PLA-PEG-NC) or the animals in the control groups (INT, DMA-PEG, and blank NC). Animals treated during the acute phase (oral route). In this work, the Y strain, which is partially resistant to BZ, was used because it induces high parasitemia levels and mortality in mice,
which are important parameters to be evaluated in parallel to therapeutic activity. Three of eight (37.5%) animals treated with free LYC presented significant reductions (P ⬍ 0.05) in patent parasitemia compared to that in the control groups (INT, DMA-PEG, and blank NC). However, this parasitemia was higher than that observed in animals treated with LYC-PCL-NC, LYC-PLA-PEGNC, and BZ (Fig. 2A). Animals treated with LYC-PCL-NC showed significantly lower parasitemia (P ⬍ 0.05) than that observed in the control groups (Fig. 2A). All animals treated with LYC-PLA-PEG-NC (5 mg/kg/ day) and BZ (100 mg/kg/day) showed total suppression of parasitemia, which became subpatent during and after treatment (Fig. 2A and Table 1). No cure was observed in animals treated with free LYC. Animals treated with LYC-PCL-NC (5 mg/kg/day) showed a 57% (4/7 animals) cure rate, and mice treated with the LYC-PLA-PEG-NC or BZ (100 mg/kg/day) formulation displayed a cure rate of 62.5% (5/8 animals). No cure was observed in the control groups (Table 2). For the groups treated with free LYC and LYC-PCL-NC, the survival rates were 75.0% (6/8 animals) and 87.5% (7/8 animals), respectively, until 6 months posttreatment, when the mice were necropsied. For the groups treated with LYC-PLA-PEG-NC and BZ, 100% survival was recorded in the same period. No significant differences (P ⬎ 0.05) in survival rates were observed between groups of animals treated with free LYC, LYC-PCL-NC, LYCPLA-PEG-NC, and BZ formulations (Fig. 2B and Table 2). The INT control group and all other control groups showed a mean survival time of 20 days (data not shown). Animals treated during the chronic phase (oral route). The group of animals treated with free LYC showed 37.5% (3/8 animals) negative HC results, compared to 90.0% (9/10 animals) for the group treated with LYC-PCL-NC, 100.0% (10/10 animals) for the group treated with LYC-PLA-PEG-NC, and 12.5% (1/8 animals) for the group treated with BZ (100 mg/kg/day). All animals
TABLE 2 Efficacy of oral treatment with lychnopholide formulations and benznidazole in mice infected with Trypanosoma cruzi Y strain and treated during the acute and chronic phases of infection Infection phase, experimental group
Dose (mg/kg/day)d
Survival for up to 6 mo (% [no. of survivors/ no. of infected animals])
Negative laboratory test result (% [no. of negative results/no. of infected animals])
Cure (% [no. of animals cured/no. of infected animals])
Acute Free LYC LYC-PCL-NC LYC-PLA-PEG-NC BZ INT DMA-PEG Blank NC
5 5 5 100 0 ⫹ ⫹
75 (6/8) 87.5 (7/8) 100 (8/8) 100 (8/8) 0 (0/8) 0 (0/8) 0 (0/8)
FBEa 37.5 (3/8) 100 (8/8) 100 (8/8) 100 (8/8) 0 (0/8) 0 (0/8) 0 (0/8)
HC/PCRb 66.7 (4/6) 25 (2/8) 25 (2/8) 25 (2/8)
ELISAc 0 (0/6) 57 (4/7) 62.5 (5/8) 62.5 (5/8)
0 (0/6) 57 (4/7) 62.5 (5/8) 62.5 (5/8) 0 (0/8) 0 (0/8) 0 (0/8)
Chronic Free LYC LYC-PCL NC LYC-PLA-PEG-NC BZ INT DMA-PEG Blank NC
5 5 5 100 0 ⫹ ⫹
70 (7/10) 100 (10/10) 90 (9/10) 80 (8/10) 70 (7/10) 70 (7/10) 70 (7/10)
HCb 37.5 (3/8) 90 (9/10) 100 (10/10) 12.5 (1/8) 0 (7/7) 0 (7/7) 0 (7/7)
PCRb 28.6 (2/7) 70 (7/10) 88.9 (8/9) 12.5 (1/8)
ELISAc 0 (0/7) 30 (3/10) 55.6 (5/9) 0 (0/8) 0 (0/7) 0 (0/7) 0 (0/7)
0 (0/7) 30 (3/10) 55.6 (5/9) 0 (0/8) 0 (0/7) 0 (0/7) 0 (0/7)
a
FBE was performed during treatment and at 30 dpt. HC and PCR were performed at 30 dpt and at 60 and 90 dpt, respectively. c ELISA was performed at 90 and 180 dpt. d ⫹, the amount of excipients used for the DMA-PEG solution and blank NC was the same as that used in the BZ and LYC-NC formulations. b
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TABLE 3 Efficacy of intravenous treatment with LYC formulations and BZ in mice with chronic infection with Trypanosoma cruzi Y strain
Experimental group
Dose (mg/kg/day)d
Survival for up to 6 mo (% [no. of survivors/ no. of infected animals])
Free LYC LYC-PCL-NC LYC-PLA-PEG-NC BZ INT DMA-PEG Blank NC
2 2 2 50 0 ⫹ ⫹
40 (4/10) 90 (9/10) 100 (10/10) 50 (5/10) 50 (5/10) 60 (6/10) 50 (5/10)
Negative laboratory test result (% [no. of negative results/no. of infected animals]) HCa
PCRb
ELISAc
Cure (% [no. of animals cured/no. of infected animals])
50 (3/6) 100 (9/9) 80 (8/10) 100 (6/6) 0 (0/5) 0 (0/6) 0 (0/5)
17.7 (1/6) 77.8 (7/9) 60 (6/10) 66.7 (4/6)
0 (0/4) 33.3 (3/9) 50 (5/10) 0 (0/5) 0 (0/5) 0 (0/6) 0 (0/5)
0 (0/4) 33.3 (3/9) 50 (5/10) 0 (0/5) 0 (0/5) 0 (0/6) 0 (0/5)
a
HC was performed at 30 dpt. PCR was performed at 60 and 90 dpt. c ELISA was performed at 90 and 180 dpt. d ⫹, the amount of excipients used for the DMA-PEG solution and blank NC was the same as that used in the BZ and LYC-NC formulations. b
(100%) of the control groups were positive by HC (Table 2). PCR was negative for 28.6% (2/7 animals) of the animals treated with free LYC, 70.0% (7/10 animals) of those treated with LYC-PCLNC, 88.9% (8/9 animals) of those treated with LYC-PLA-PEGNC, and 12.5% (1/8 animals) of those treated with BZ (Table 2). All animals treated with free LYC or BZ and all animals in the control groups (INT, DMA-PEG, and blank NC) were ELISA positive for anti-T. cruzi IgG. ELISA was negative for 30% (3/10 animals) of the animals treated with LYC-PCL-NC and 55.6% (5/9 animals) of those treated with LYC-PLA-PEG-NC (Table 2). Considering the cure criterion adopted, no cure was observed in animals treated with free LYC or BZ or in the control groups (Table 2). Animals treated with LYC-PCL-NC and LYC-PLAPEG-NC showed cure rates of 30% (3/10 animals) and 55.6% (5/9 animals), respectively (Table 2). Animal survival rates for up to 6 months posttreatment are illustrated in Table 2. The percentages of survival for the groups of mice treated with different formulations were as follows: free LYC, 70% (7/10 animals); LYC-PCL-NC, 100% (10/10 animals); LYCPLA-PEG-NC, 90% (9/10 animals); and BZ, 80% (8/10 animals) (Table 2). The untreated control groups (INT, DMA-PEG, and blank NC) all showed a survival rate of 70% (7/10 animals) (Table 2). Animals treated during the chronic phase (intravenous route). For the group of animals treated with free LYC (2 mg/kg/ day), 50% (3/6 animals) were negative by HC, compared to 100% (9/9 animals) of the animals treated with LYC-PCL-NC, 80% (8/10 animals) of those treated with LYC-PLA-PEG-NC, and 100% (6/6 animals) of those treated with BZ. All animals of the control groups were positive by HC (Table 3). PCR was negative for 17.7% (1/6 animals) of the animals treated with free LYC, 77.8% (7/9 animals) of those treated with LYC-PCL-NC, 60% (6/10 animals) of those treated with LYCPLA-PEG-NC, and 66.7% (4/6 animals) of those treated with BZ (Table 3). All animals treated with free LYC or BZ (50 mg/kg/day) or in the control groups were ELISA positive. ELISA was negative for 33.3% (3/9 animals) of the animals treated with LYC-PCL-NC and 50% (5/10 animals) of the animals treated with LYC-PLAPEG-NC (Table 3). No cure was observed in the animals of the free LYC, BZ, and control groups (Table 3). Considering the cure criterion adopted, animals treated with LYC-PCL-NC and LYC-PLA-PEG-NC showed cure rates of 33.3% (3/9 animals) and 50% (5/10 animals), respectively (Table 3).
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Animal survival rates for up to 6 months posttreatment are illustrated in Table 3. The survival rates for animals treated with the different formulations were as follows: free LYC, 40% (4/10 animals); LYC-PCL-NC, 90% (9/10 animals); LYC-PLA-PEGNC, 100% (10/10 animals); and BZ, 50% (5/10 animals). The untreated control (INT) group and the blank NC group both showed a survival rate of 50% (5/10 animals), similar to that observed for the DMA-PEG group (60% [6/10 animals]). DISCUSSION
Despite the large number of new tested compounds that have shown anti-T. cruzi activity in vitro and efficacy in vivo, only four molecules have been subjected to clinical trials for CD. In general, the results with these drugs were contradictory or even unsatisfactory (7, 35, 36). In an attempt to overcome the limitations of CD therapy, some researchers recently suggested the development of drug delivery systems using nanocarriers to improve the drug pharmacological effects (35, 37) by controlling drug release, reducing toxicity, and modulating the pharmacokinetics and biodistribution (8, 35, 37, 38). Few studies have investigated the association of drugs or chemical leads with polymeric nanocarriers for the treatment of CD. D0870, a bis-triazole compound, was able to improve the cure rate to 60% for animals infected with the Y strain (38). Other examples are the association of BZ with self-emulsifying delivery systems (SNEDDS), which increased the index of cure for Swiss mice infected with the same T. cruzi strain (35). Furthermore, the natural isolated compound lychnopholide (LYC) associated with polymeric NC was highly efficacious against T. cruzi infection in mice (18). A recent study with rats suggested the favorable use of oral LYC as an active substance or pharmacological agent because of its physicochemical properties of good permeability and rapid absorption, distribution, and elimination, resulting in 68% oral bioavailability (21). However, LYC has poor solubility, and its fast elimination implies its possible administration in repeated doses. Controlled-release formulations, such as NC, may improve the pharmacokinetics and biodistribution and thus maintain efficacy with fewer doses. For our studies, we selected the murine model because it is considered the most susceptible animal model for experimental T. cruzi infection and is the most widely used model for screening of drugs for CD (29, 39). A recent experience of our group in the intravenous treatment of Swiss mice infected with the Y strain of T. cruzi with 2 mg/kg/
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day of LYC in NC for 20 consecutive days resulted in significantly better efficacy than that with free LYC or BZ (18). Considering these previous results and the importance of the oral route of administration for potential compounds against chronic CD, we decided to compare in the present study the oral administration of LYC-NC formulations for the treatment of the acute and chronic phases of infection in mice infected with the T. cruzi Y strain with the results obtained for the chronic phase upon intravenous administration of the drug. In order to avoid toxicity, the dose of 5 mg/kg was chosen for oral administration. The NC formulations (LYC-PLA-PEG-NC and LYC-PCLNC) presented a bluish milky aspect characteristic of colloidal suspensions. The experimental average particle diameters of the formulations, ranging from 106 to 190 nm, and a low polydispersity index (40) warrant safety by intravenous administration and a low risk of embolic events. LYC-PLA-PEG-NC showed reduced sizes compared to those of LYC-PCL-NC (18). The smaller size of LYC-PLA-PEG-NC could be attributed to a difference in the arrangement of the hydrophilic block of the PEG polymer on the NC surface, which influences interfacial tension between solvents during their mixing, generating particles of smaller size. Moreover, NC of small size are more prone to have access to infectious and inflammatory foci due to their ability to diffuse through the leaky endothelium (41). This aspect may be particularly interesting for CD treatment. The oral administration of LYC at 5 mg/kg/day loaded in PLAPEG-NC during the acute phase in mice infected with T. cruzi strain Y led to a higher efficacy than those for all other groups. LYC-PCL-NC and BZ also led to significant parasitemia reductions compared to those for the free LYC and control groups. The survival of infected animals treated with LYC-PLA-PEG-NC and BZ was total and higher than those for the groups treated with LYC-PCL-NC and free LYC, but survival was null for the control groups. In addition, the cure rates observed were higher with LYC-PLA-PEG-NC (62.5%) and BZ (62.5%) and similar to that with LYC-PCL-NC (57%). However, our previous results showed higher cure rates (18) when animals infected with the same T. cruzi strain were treated via intravenous administration with the same LYC-NC formulations during the acute phase of the infection. A higher percentage of cure upon oral administration during the chronic phase of Y strain infection was obtained with LYCPLA-PEG-NC (55.6%) than with LYC-PCL-NC (30%), while animals in all other groups (free LYC, BZ, and controls) were not cured. Intravenous administration of the same set of formulations at a different dose (2 mg/kg/day) and under the same conditions of treatment during the chronic phase of the infection revealed cure rates of 50.0% with LYC-PLA-PEG-NC and 33.3% with LYCPCL-NC. No cure was observed for animals of the other groups (free LYC, BZ, and controls). Therefore, sterically stabilized NC, such as LYC-PLA-PEG-NC, were found to improve the cure rates of LYC, in agreement with the previously reported improvement in efficacy observed for T. cruzi-infected mice in the acute phase (18). Thus, for the chronic phase, similar indexes of cure were reached between intravenous (50% and 33.3%) and oral (55.6% and 30%) treatments for LYC-PLA-PEG-NC and LYC-PCL-NC, respectively. All experiments in the present work and previous reports by our team (18) revealed that the administration of LYC loaded in NC formulations provided higher cure rates than those for treatment with LYC in solution, regardless of the route of administration, dose, and period of the infection when treatment
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was initiated. It was again demonstrated that free LYC reduced parasitemia and increased the survival rate of mice compared to those for control groups, although none of the infected and treated animals were healed, which shows the therapeutic advantage and effectiveness of encapsulation of LYC in polymeric NC. The survival rates observed for the control groups in the chronicphase model (INT, blank NC, and DMA-PEG solution) were expected, because these animals were infected with an inoculum 20 times lower than that for mice infected for treatment of the acute phase, a condition necessary to enable chronic infection using the T. cruzi Y strain. The kinetic release profile of LYC from NC was already determined (14). The time required to release 50% (T50) of LYC from LYC-PCL-NC was found to be 20.7 h, versus a dissolution T50 of 7.3 h for LYC powder in release medium. Thus, the improved efficacy observed with LYC-PCL-NC compared to free LYC may be due to the slow release of LYC from NC, i.e., a form of sustained release. In general, the PEG corona surrounding NC reduces the rate of release of lipophilic drug to the external medium in the presence of proteins present there, as previously reported (42, 43). Furthermore, PEG prevents the access of proteins to the NC oily core, which may reduce LYC release or NC wall degradation. The significantly higher cure rates obtained with the PEG-containing formulation (LYC-PLA-PEG-NC) than with the conventional formulation (LYC-PCL-NC) by the oral route can thus be interpreted as a consequence of different rates of release and/or the additional protection against the GIT medium and the action of enzymes provided by PEG at the surface (44, 45). The differences between the cure results we observed with the sterically stabilized and conventional NC formulations are in agreement with findings reported in the literature for other drugnanocarrier systems (18, 44, 46, 47). Nanoparticles of PEG have the ability to extend the circulation time due to reduction of opsonization and avoidance of subsequent clearance by the mononuclear phagocytic system (MPS). Consequently, nanoparticles are not eliminated or metabolized as fast as free LYC and may accumulate and act in inflamed tissues, as occurs in T. cruzi infection. This may be the main cause of the improved efficacy of LYC loaded in NC. Although the oral route is the route most used for drug administration, some drawbacks are found for active chemical entities, such as LYC, that suffer degradation at extreme pH. The higher cure rate for animals infected with the Y strain of T. cruzi observed here upon oral administration of encapsulated LYC is an indication that the effect of the molecule was preserved, which in turn is an indication that the molecular integrity was maintained. Polymeric nanocarriers have the ability to interact with infected target cells via a variety of endocytic mechanisms (35) and to deliver anti-T. cruzi drugs or substances inside the cell (35, 37, 38). In the acute phase of infection, the therapeutic strategy consists of maintaining the nanoparticles in the blood due to the large number of trypomastigotes in this compartment. This can be achieved with the use of either type of NC studied here, as they both circulate longer in the blood compartment than free LYC does. On the other hand, during the chronic phase of infection, the strategy is to reach the amastigote forms of the parasite inside the cell by using small, long-circulating nanocarriers (NC) and also to reach the interstitial matrix, where the amastigotes or trypomastigotes derived from them are released. This approach takes
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Oral Lychnopholide Nanocapsules Cure Chagas Disease
advantage of the endothelial leakage of inflamed tissues, which results in nanoparticle perfusion to the extravascular region (35). In conclusion, this work is the first report of cure of experimental Chagas disease via oral administration of an isolated natural substance loaded in sterically stabilized and conventional polymeric nanocapsules in both phases of infection with a T. cruzi strain partially resistant to benznidazole. Such levels of cure have never been achieved for chronic experimental Chagas disease with a T. cruzi strain partially resistant to treatment. These findings represent a new and important perspective for treatment of Chagas disease by the oral route.
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ACKNOWLEDGMENTS We thank G. Pound-Lana for English grammar revisions. We thank also the UFOP and CAPES undergraduate and postgraduate scholarships. We have no conflicts of interest to declare.
FUNDING INFORMATION
18.
This work, including the efforts of Dênia Antunes Saúde-Guimarães, Vanessa Carla Furtado Mosqueira, and Marta de Lana, was funded by FAPEMIG as NANOBIOMG (00007-14 and 40/11), TOXIFAR (0043213), and BIOTERISMO (APQ 00432-12) networks and projects (APQ00566-11, APQ-00956-13) and partially funded by CAPES-COFECUB 768/13. Marta de Lana and Vanessa Carla Furtado Mosqueira are research fellows of Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/MCTI).
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