Intracellular Bacteria and Protozoa - Intracellular Delivery: Fundamentals and Applications - page 789

First strategies were only tested in vitro . Stearylamine liposomes showed
activity against epimastigotes (only found in bugs), trypomastigotes and axenic
amastigotes; these liposomes however, were not assayed against amastigotes in
infected cells, the most relevant target of the disease (Yoshihara et al. 1987 ). The
same occurred with alkylpolycyanoacrylate nanospheres containing nifurtimox
(Gonzalez-Martin et al. 1998 ; Sánchez et al. 2002 ) and allopurinol (Gonzalez-
Martín et al. 2000 ), that showed some activity against epimastigotes and intracel-
lular amastigotes, although empty nanospheres showed also trypanocidal
activity.
The activities of four AmB formulations, Fungizone, AmBisome, Amphocil and
Abelcet, were compared in vitro and in vivo on an acute murine model infected
with the Y and the Tulahuen strains (Yardley and Croft 1999 ). When tested against
trypomastigotes of the Y strain upon 24 h incubation, Fungizone showed the highest
activity (LD 50 0.3 mg/ml), followed by Amphocil (LD 50 between 1 and 0.5 mg/ml).
Abelcet and AmBisome did not show activity up to a 30 mg/ml. When tested
against amastigotes infecting murine peritoneal macrophages, Fungizone and
Amphocil showed the highest activities (IC 50 between 0.027 and 0.028 mg/ml), fol-
lowed by AmBisome, Abelcet and benznidazole, with IC 50 of 0.19, 1.2 and
1.43 mg/ml, respectively. On amastigote-infected Vero cells, Fungizone, Amphocil,
AmBisome, Abelcet and benznidazole IC 50 increased to 2, 4.1, 3.6, 2.3 and 4.2 mg/ml,
respectively.
The effect of multiple and single doses of amphotericine B doxycholate and
liposomal amphotericine (AmBisome) on Balb/c mice infected with Y and
Tulahuen strain was also evaluated. On T. cruzi Y strain-infected mice, the admin-
istration of six doses of Fungizone (0.5 mg/kg, maximum tolerated dose) or
AmBisome (12.5 mg/kg) enabled 4 of 5 and 5 of 5 mice to survive until the end of
the experiment (60 days post-infection). The effect of AmBisome was not curative
since trypomastigotes were observed in the tail blood of treated mice for 3 weeks
post-treatment. Untreated control animals died 18 days post-infection. On T. cruzi
Tulahuen strain-infected mice, the lowest dose of AmBisome (6.25 mg/kg) pro-
duced survival of all animals until 60 days post-infection, meanwhile untreated
controls survived until 13 days post-infection.
Used as a single dose at 25 mg /kg AmBisome induced the survival of 5 of 5
mice, whereas Abelcet and Amphocil and five consecutive oral doses of benznida-
zole at 45 mg/kg only induced the survival of 3/5 at the end of 60 days (lower doses
failed).
In general terms, in vivo AmBisome was more effective and less toxic than
other lipid amphotericine formulations, despite of a delayed elimination of blood
parasites when compared to benznidazole (3 vs. 1 week). A crucial difference
between the use of AmBisome against leishmaniasis (a parasite that colonizes the
phago-lysosomal system in cells of the RES) and against Chagas is that in the lat-
ter, even if properly biodistributed, the intracellular pathway followed by amphot-
ericine could not be appropriate. It is possible that once inside the target cell, the
hydrophobic nature of the amphotericine impaired its diffusion from the endo/
phago-lisosomal confinement.