Cure of experimental Chagas' disease by the bis-triazole DO870 incorporated into ‘stealth’ polyethyleneglycol–polylactide nanospheres

Judith Molinaa,b, Julio Urbinac, Ruxandra Grefd, Zigman Brenera and José Maciel Rodrigues Júniore,*

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
We have incorporated several inhibitors of sterol biosynthesis into long-circulating polyethyleneglycol–polylactide (PEG–PLA) nanospheres in order to improve the bioavailability of these poorly soluble compounds. Mice infected with CL and Y strains of Trypanosoma cruzi and treated for 30 consecutive days with DO870-loaded nanospheres at doses of 3 mg/kg/day, by the intravenous route, showed a significant cure rate (60–90%) for both strains. The activity was dose dependent and significant activity was observed for doses >= 0.75 mg/kg/day. No cure was observed in mice treated with unloaded nanoparticles. Ketoconazole and itraconazole failed to induce cure against the Y strain even in the entrapped form.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Only two drugs that are active against Trypanosoma cruzi, nifurtimox and benznidazole, have reached the pharmaceutical market in the last decades and have been used in chagasic patients.1 Although designed for the treatment of diseases not related to Chagas' disease, many drugs have fortuitously exhibited activity against T. cruzi. Current developments in experimental Chagas' disease have highlighted the potential of sterol biosynthesis inhibitors (SBIs).2 Their specific activity is based on the depletion of essential endogenous sterol or on the accumulation of toxic intermediates or both.2

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-{epsilon}-caprolactone (PCL) and polyethyleneglycol (PEG–PLA, PLGA–PEG, PCL–PEG) 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 PEG–PLA nanospheres and the evaluation of this new drug delivery system against infections caused by two strains of T. cruzi in an experimental mouse model.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The experimental protocols were carried out with two strains of T. cruzi: the nitroimidazoles/nitrofurans-susceptible strain CL, isolated from naturally infected Triatoma infestans and the Y strain, isolated from a patient in acute phase, which displays intermediate resistance to these drugs. The original isolates were maintained as trypomastigotes in liquid nitrogen, periodically transferred to mice and refrozen, with full retention of their biological and drug resistance characteristics. Handling of live T. cruzi was according to established guidelines. Groups of 10–15 Swiss albino female mice (from Fiocruz Animal Facilities, Rio de Janeiro, Brazil), weighing 18–20 g, were inoculated ip with 104 blood trypomastigotes. Drugs were administered daily during the acute phase of the disease, i.e. starting 4 days post-infection (p.i.) and continued for 30 days. Free drug suspensions were administered by the oral route and drug-loaded nanoparticles and unloaded nanoparticles were administered iv by the tail vein. Untreated mice were used as controls. The mortality rate was recorded daily. In order to verify a parasitological cure, haemoculture, xenodiagnosis and detection of anti-live T. cruzi antibodies were determined in surviving animals. The death of untreated animals started 7–10 days p.i. and by 50 days p.i. all untreated mice were dead.

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 PEG–PLA nanospheres were administered by the intravenous route. The drugs incorporated into nanospheres were ketoconazole, itraconazole and DO870. PEG5K–PLA45K (PEG–PLA), 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 ultrafiltration–centrifugal 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 Kaplan–Meier non-parametric method was used to estimate the survival functions of the different experimental groups and rank test (log-range and Peto–Peto–Wilcoxon) was used to compare them.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The unloaded and drug-loaded nanoparticles had a monodisperse size distribution between 100 and 200 nm. The efficiencies of drug entrapment were 90, 87 and 92% for DO870, itraconazole and ketoconazole, respectively. Benznidazole was not incorporated into nanoparticles because its physico-chemical characteristics are not compatible with this carrier. The method is reproducible and the stability of the formulations was optimal at 4°C and they retained their physical and chemical characteristics during the study.

Nanoparticles were well tolerated by infected mice since no signal of acute toxicity was observed after 30 iv injections. As shown in Table IGo, mice infected with CL strain and treated with DO870 by the oral (5 mg/kg/day) or iv (1.5–3.0 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).


View this table:
[in this window]
[in a new window]
 
Table I. Activity of DO870 and DO870-loaded PEG–PLA nanospheres in a murine model of short-term CL strain Chagas' diseasea
 
Based on these previous data the experiments against strain Y (partially resistant to benznidazole and nifurtimox) were carried out with intravenous administration of entrapped DO870 at doses of 3 mg/kg/day (Table IIGo). The survival of the animals treated with entrapped DO870 at 3 mg/kg/day for 30 days was comparable to that of mice receiving benznidazole at 100 mg/kg/day. Both groups displayed a statistically significant survival (P < 0.0001) when compared with the untreated control group. On the other hand, the survival of animals treated with ketoconazole- or itraconazole-loaded nanospheres (3 mg/kg/day) was not significantly different from that of the untreated control group.


View this table:
[in this window]
[in a new window]
 
Table II. Activity of benznidazole and DO870-, itraconazole- and ketoconazole-loaded PEG–PLA nanospheres in a murine model of short-term Y strain Chagas' diseasea
 
Table IIGo indicates that entrapped DO870 at 3 mg/kg/day induced a 60% cure. The group that received benznidazole at 100 mg/kg/day showed a 47% cure. No parasitological cure was obtained with itraconazole- or ketoconazole-loaded nanospheres. Previous studies have described that ketoconazole and itraconazole failed in the cure of these strains when administered orally.1,10 In our study these drugs also failed when administered intravenously. Since the drugs studied are totally released from nanoparticles (data not shown), the pharmacokinetic profiles must be clarified to understand their inefficacy.

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 15–20 mg/kg/day or every other day for a total of 28 doses was able to cure 70–90% 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.


    Acknowledgments
 
We thank Dr Olindo Martins-Filho for his assistance during studies of flow cytometry. This work received financial support from FAPEMIG, CNPq, PRPq-UFMG, PRONEX, CONICIT and the UNDP World Bank/World Health Organization Program for Research and Training in Tropical Diseases (grant 970297).


    Notes
 
* Correspondence address. Faculdade de Farmácia, Universidade Federal de Minas Gerais, Av. Olegário Maciel, 2360 CEP: 30180-112, Belo Horizonte (MG), Brazil. Fax: +55-31-291-97-69; E-mail: rodrigue{at}dedalus.lcc.ufmg.br Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Brener, Z., Cançado, J. R., Galvão, L. M. C., Luz, Z. M. P., Filardi, L. S., Pereira, M. E. S. et al. (1993). An experimental and clinical assay with ketoconazole in the treatment of Chagas' disease. Memórias do Instituto Oswaldo Cruz 88, 149–53.

2 . Urbina, J. A. (1999). Chemotherapy of Chagas' disease: the how and the why. Journal of Molecular Medicine 77, 332–8.[ISI][Medline]

3 . Urbina, J. A., Payares, G., Molina, J., Sanoja, C., Liendo, A., Lazardi, K. et al. (1996). Cure of short- and long-term experimental Chagas' disease using D0870. Science 273, 969–71.

4 . 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, 137–40.[Abstract/Free Full Text]

5 . Melo, R. C. & Brener, Z. (1978). Tissue tropism of different Trypanosoma cruzi strains. Journal of Parasitology 64, 475–82.[ISI][Medline]

6 . Rodrigues, J. M., Croft, S. L., Fessi, H., Bories, C. & Devissaguet J. P. (1994). The activity and ultrastructural localization of primaquine-loaded poly(d,l-lactide) nanoparticles in Leishmania donovani infected mice. Tropical Medicine and Parasitology 45, 223–8.[ISI][Medline]

7 . Gref, R., Minamitake, Y., Peracchia, M. T., Trubetskoy, V., Torchilin, V. & Langer, R. (1994). Biodegradable long-circulating polymeric nanospheres. Science 263, 1600–3.[ISI][Medline]

8 . Filardi, L. S. & Brener, Z. (1987). Susceptibility and natural resistance to Trypanosoma cruzi strains to drugs used clinically in Chagas' disease. Transactions of the Royal Society of Tropical Medicine and Hygiene 81, 755–9.[ISI][Medline]

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, 569–73.[Abstract]

10 . Araújo, M. S., Molina, J., Pereira, M. E. S. & Brener, Z. (1996). Combination of drugs in the treatment of experimental Trypanosoma cruzi infection. Memorias do Instituto Oswaldo Cruz 91, Suppl. 1, 315.

Received 8 February 2000; returned 11 May 2000; revised 5 July 2000; accepted 18 August 2000