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INTRODUCTION: The impact of cyclosporine on hepatocyte
apoptosis is controversial. In vitro, cyclosporine is known to block the mitochondrial permeability transition observed during the apoptotic process. Cyclosporine, which is very lipophilic, requires adequate solubilization with agents such as Cremophor ® EL (CrEL). However, CrEL has been recognized to be associated with side effects and is also known for its capacity to inhibit the multidrug resistance p-glycoprotein (as cyclosporine does). We have previously reported that an iv-formulation of CsA protected against Fas-induced apoptosis in the liver. The following studies were performed to assess the effects of CrEL, the vehicle used in the cyclosporine formulation, on Fas-induced liver injury in vivo. METHODS: BALB/C mice were injected intraperitoneally (ip) with 0.45 μg/g anti-Fas antibody,
a dose that induces acute liver injury after 5-6 hours without animal demise. The formulation-vehicle, (CrEL/Ethanol(EtOH)) was injected ip 45 min prior to anti-Fas. Animals were sacrificed at six time points up to 7.5hrs following anti-Fas. Standard enzymatic assays were used for serum-ALT/AST and caspase-3 determinations. tBID/BID was evaluated by Western blot and GSH/GSSG by HPLC.RESULTS: Induction of hepatocellular injury in vivo by anti-Fas led to a characteristic sequence of biochemical
events. The appearance of tBID (35.3±4.9 BID) and
induction of Caspase-3 activity (26.8±5.8 U/L) were first
observed 3h post-injection. Pre-treatment with CrEL/EtOH abolished the increases in tBID (6.8±2.0; P<0.001 vs. Fas-only) and Caspase-3 activity (3.8±2.2 U/L; P<0.001 vs. Fas-only). Five hours post-anti-Fas, serum-ALT/AST levels rose to 7584±2419/5369±1158 U/L (Fas-only) and intracellular glutathione (GSH) were decreased (34.6±7.5 control, P<0.001 vs. control). This was associated with the appearance of massive liver injury as shown on histology. CrEL/EtOH significantly attenuated the increase in serum-ALT/AST (895±474/1230±448 U/L; P<0.05 vs. Fas-only) as well as the decrease in GSH levels (86.6±3.9 control; P<0.001 vs Fasonly). Morphological analysis also supported the effect of CrEL/EtOH on Fas-induced apoptosis. CONCLUSION: Our results demonstrate a protective effect of the vehicle used in the intravenous formulation of cyclosporine on all levels of the injury process studied in Fas-induced hepatocyte apoptosis. Experiments are underway to determine the mechanisms responsible for this observation. These results might explain the discrepancy observed in some of the previous experiments performed with cyclosporine. They also underline the importance of the choice of vehicle in drug development.
