a Laboratório de Doença de Chagas, Centro de Pesquisas René Rachou, Belo Horizonte, Brazil; b Departamento de Parasitología, Instituto de Zoología Tropical, Universidad Central de Venezuela, Caracas 1041, Venezuela; c Laboratorio de Química Biológica, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas, Apartado 21827, Caracas 1020 A, Venezuela; d UMR CNRS 8612, Faculté de Pharmacie Chatenay-Malabry, France; e Laboratório de Tecnologia Farmacêutica, Departamento de Produtos Farmacêuticos, Universidade Federal de Minas Gerais, Brazil
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Abstract |
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Introduction |
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The fourth-generation azole derivatives (inhibitors of fungal cytochrome P450-dependent C14 sterol demethylase), such as DO870, are capable of inducing parasitological cure in murine models of both acute and chronic Chagas' disease.3 DO870 is also able to promote the cure of infection caused by nifurtimox- and benznidazole-resistant strains.4 Although this drug has shown promising activity against Chagas' disease, the development of this compound has recently been discontinued.2
The search for alternatives in the treatment of microbial and parasitic diseases has pointed out the potential of drug targeting with colloidal systems (e.g. liposomes and nanoparticles). In the specific case of Chagas' disease, few studies have been carried out, probably because of the disseminated localization of the parasite.5 In fact, the metacyclic trypanosomes evade the immune system by invading a variety of cell types, including muscles and the gastrointestinal tract, as well as those of the mononuclear phagocyte system (MPS). This makes the application of the conventional carriers, which are designed to target the MPS difficult.6 Polymeric nanoparticles have been proposed for passive drug delivery to macrophages because of their rapid clearance from the plasma by the MPS. The search for new polymers has led to the development of polylactide (PLA) polymers and co-polymers, which are biocompatible and are hydrolysed slowly to innocuous products. The recent development of nanoparticles prepared with a copolymer of PLA or polylactic-co-glycolic acid (PLGA) or poly--caprolactone (PCL) and polyethyleneglycol (PEGPLA, PLGAPEG, PCLPEG) for controlled iv delivery of drugs has shown their ability to avoid opsonization and uptake by the MPS (stealth' nanoparticles), increasing their plasma circulation time and consequently, the half-life of entrapped drugs in the plasma.7
Based on these new concepts, the aim of this work was the development of an injectable formulation of SBIs trapped in PEGPLA nanospheres and the evaluation of this new drug delivery system against infections caused by two strains of T. cruzi in an experimental mouse model.
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Materials and methods |
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The drugs used for the treatment of experimental Chagas' disease were DO870 (a gift from Dr John Ryley of Zeneca Pharmaceuticals, Macclesfield, UK), ketoconazole and itraconazole (Janssen Pharmaceutics, São Paulo, Brazil) and benznidazole (Roche, São Paulo, Brazil). The drugs given orally by gavage were suspended in aqueous 2% methylcellulose plus 0.5% Tween 80 and the drugs incorporated into PEGPLA nanospheres were administered by the intravenous route. The drugs incorporated into nanospheres were ketoconazole, itraconazole and DO870. PEG5KPLA45K (PEGPLA), was synthesized by ring opening polymerization of lactide (Aldrich, Milwaukee, NI, USA) initiated by the hydroxyl end group of monomethoxy polyethyleneglycol (MPEG) (Sigma, St Quentin, France) with an average molecular weight of 5000 using stannous octoate as catalyst in equimolar quantity with regard to MPEG. The polymer was characterized by size exclusion chromatography coupled to a refractive index and a multiangle light scattering detector (MALLS, Wyatt Dawn model F, Wyatt Technology Corp., Milton Roy, USA). The average molecular mass was 45 kDa with a polydispersity index of 1.2. 1H NMR confirmed these results. The nanoparticles were prepared by the simple emulsification method.8 Polymer (25 mg) and drugs (7.5 mg) were dissolved in 2 mL of methylene chloride and emulsified in 15 mL of sterile water for injection using a sonifier (Branson Sonifier 250). The organic solvent was eliminated by evaporation under gentle stirring. The diameter of the particles was determined by laser scattering using a Cilas apparatus. Quantification of DO870, itraconazole and ketoconazole was conducted using a Hitachi spectrophotometer and the wavelengths were 291, 263 and 246 nm and showed extinction coefficients of 32847, 35282 and 31108, respectively. The entrapment rate was determined after dissolution of the particles in acetonitrile. Free drug was quantified after an ultrafiltrationcentrifugal method using a unit filter system (Millipore). In order to verify the occurrence of parasitological cure of the short-term disease in surviving animals, haemoculture, xenodiagnosis and detection of anti-live T. cruzi antibodies were evaluated.2,4,9 Parasitaemia was measured in tail blood with a haemocytometer. Haemocultures and xenodiagnosis were carried out as described previously.10 Antibodies against live T. cruzi were evaluated by cytofluorometry, using the procedure of Martins-Filho et al.,9 with minor modifications. Fisher's test was applied to evaluate the significance of the results for cure rate analysis. The KaplanMeier non-parametric method was used to estimate the survival functions of the different experimental groups and rank test (log-range and PetoPetoWilcoxon) was used to compare them.
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Results and discussion |
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Nanoparticles were well tolerated by infected mice since no signal of acute toxicity was observed after 30 iv injections. As shown in Table I, mice infected with CL strain and treated with DO870 by the oral (5 mg/kg/day) or iv (1.53.0 mg/kg/day) route showed no mortality up to 93 days after infection, while in the untreated control group, 100% death was observed within 93 days p.i. Only the control group and the group that received the lowest dose of DO870 (0.75 mg/kg/day) were positive to haemoculture and xenodiagnostic tests 110 days p.i. In this treated group the cure rate was 43%. No reappearance of circulating parasites occurred in animals treated with doses
1.5 mg/kg/day iv. In these groups the cure rate was 70 and 90% for the doses of 1.5 and 3 mg/kg/day, respectively. These results are comparable to those obtained with oral administration of DO870 5.0 mg/kg/day where 80% of infected mice were cured (without significant difference, P < 0.05). Unloaded nanoparticles showed no activity against T. cruzi when administered by the iv route (data not shown).
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Intravenous injectable particles are generally eliminated by the MPS, accumulating in the liver and spleen within a few minutes. The presence of a hydrophilic coating that might prevent opsonization and subsequent recognition by the macrophages enables the particles to avoid the MPS and increases the circulating time of drugs in the blood.5 These sterically stabilized nanospheres may be useful in the treatment of systemic diseases including parasitic infections. The high drug entrapment rate permitted a formulation that showed effective therapeutic activity for DO870 against acute experimental Chagas' disease at doses lower than 1.0 mg/kg/day. Entrapped DO870 also showed significant activity against the virulent Y strain at a dose of 3.0 mg/kg/day, giving a cure rate of 60%. In comparison, the same drug given orally at 1520 mg/kg/day or every other day for a total of 28 doses was able to cure 7090% of mice infected with the same strain, as described previously by Urbina and collaborators.3 Indeed, the pharmacokinetic profile of this drug was not linear when given orally.2 Our results also showed a dose-dependent efficacy against a CL strain after iv administration. This new formulation may provide a better pharmacokinetic profile, since the nanoparticles are eliminated slowly from the circulation, probably providing a sustained drug release.
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Acknowledgments |
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Notes |
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References |
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Molina, J., Brener, Z., Romanha, A. J. & Urbina, J. A. (2000). In vivo activity of the bis-triazole DO870 against drug-susceptible and drug-resistant strains of the protozoan parasite Trypanosoma cruzi. Journal of Antimicrobial Chemotherapy 46, 13740.
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9 . Martins-Filho, O. A., Pereira, M. E., Carvalho, J. F., Cançado, J. R. & Brener, Z. (1995). Flow cytometry, a new approach to detect anti-live trypomastigote antibodies and monitor the efficacy of specific treatment in human Chagas' disease. Clinical and Diagnostic Laboratory Immunology 2, 56973.[Abstract]
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Received 8 February 2000; returned 11 May 2000; revised 5 July 2000; accepted 18 August 2000