Biomedical Engineering Reference
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To minimize opsonization, RES uptake and to act as a long circulating drug
depot, different azoles (itraconazole, ketokonazole and the fourth generation bis-
triazole D0870) were loaded in PEG-co-PLA nanospheres. Nanoparticles were daily
i.v. administered to swiss albino mice infected with a nitroimidazoles/nitrofurans-
susceptible and a partially resistant strain, for 30 days (Molina et al. 2001 ). D0870
in nanospheres (3 mg/kg) produced similar percentages of cure (90%) than free
D0870 (30 oral doses at 5 mg/kg) (80%) on the susceptible strain. In the case of the
resistant strain, D0870 containing nanospheres (60% cure) were more effective than
itraconazole- and ketokonazole-nanospheres (0% cure) and 30 daily oral doses of
benznidazole (100 mg/kg) (47%). However, 20 oral doses of free D0870 (5 mg/
kg day) in the same animal model produced 100% cure (Molina et al. 2000 ).
Aiming to increase the efficacy of benznidazole as trypanocidal agent by modi-
fying its pharmacokinetics and biodistribution, our research group developed a
MLV formulation of benznidazole (hydrogenated soyPC: chol: distearoyl-
phosphatidylglycerol, 2:2:1 M ratio) (Morilla et al. 2002 ). When i.v. administered
in rats as a 0.2 mg/kg, a three-fold higher benznidazole accumulation in the liver
than the achieved with the same dose of the free drug was found. However, lipo-
somal benznidazole (i.v., 0.4 mg/kg, twice-a-week from day 5 to day 22 post infec-
tion) did not decrease parasitemia levels in mice infected with the RA strain. These
results indicated that the relationship between the increased liver delivery and the
therapeutic effect of liposomal benznidazol was not that simple. The hydrophobic
benznidazole remained associated to the liposomal bilayer that kept trapped within
the endo-lysosomal pathway, instead of being released to the cell cytoplasm
(Morilla et al. 2004 ).
Acid media trigger a phase transition in pH sensitive liposomes, from bilayer at
neutral or alcaline media, to inverted hexagonal phase II when the pH drops below
5-6. The hexagonal phase II is responsible for the fusion of liposome with the endo-
lysosomal bilayer; as a result, the aqueous content of pH sensitive liposomes is
released to cell cytoplasm. The hydrophobic nature of benznidazole made it unsuit-
able for delivery from pH-sensitive liposomes, and had to be replaced by a hydro-
philic compound with trypanocydal activity. Hence, pH-sensitive liposomes (DOPE:
cholesteryl hemisuccinate, 6:4, mol:mol, ~400 nm) containing the hydrophilic
etanidazole were prepared (Morilla et al. 2005 ). This formulation ensured a fast and
massive delivery of etanidazole into the cytosol of murine J774 macrophages.
Liposomes containing etanidazole were phagocytosed by both uninfected and
T. cruzi -infected macrophages and at 200 mg/ml showed 72% anti-amastigote activ-
ity in J774 cells after 2 h. Contrary to this, the same dose of free etanidazole ren-
dered 0% activity. I.v. administration of etanidazole loaded in pH-sensitive liposomes
(0.56 mg/kg, starting 5 day post infection, 3 days-a week over 3 weeks) resulted in
a significant decrease in parasitemia (days 12, 19, 21 and 23) of Balb/c mice infected
with 50 trypomastigotes of the RA strain. Administration of a 180-fold higher dose
of free etanidazole failed to reduce the number trypomastigotes in blood.
Remarkably, previous in vitro determinations of free etanidazole trypanocidal
activity showed that on RA trypomastigotes, the LD 50 of etanidazole was 18 mM
(8.2-fold less active than benznidazole). In the case of amastigote-infected Vero
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