142. Molecular and Macromolecular Alterations of Recombinant Adenoviral Vectors Do Not Eliminate Changes in Hepatic Drug Metabolism

Abstract

The hepatic cytochrome P450 (CYP) enzyme system is responsible for the inactivation and clearance of numerous substrates and the conversion of drugs to their active metabolite(s). We have previously shown that a single dose of recombinant adenovirus (Ad) ranging from 5.7|[times]|106|[ndash]|5.7|[times]|1012 viral particles/kilogram (vp/kg) significantly alters hepatic CYP isoforms 3A2 and 2C11 for 14 days (Callahan et al, JPET, 2005, 312:492|[ndash]|501). We also found that the expression and function of these isoforms, selected for their predominance in drug metabolism (CYP3A2) and their responsiveness to inflammatory mediators (CYP2C11), is altered by certain transgene cassettes (Callahan et al, Mol. Ther., 2003, 7:S58). In an effort to determine the effect of viral gene expression and the innate immune response against virus capsid proteins on CYP, Sprague-Dawley rats were given 0.5mL (5.7|[times]|1012 vp/kg) of either: wild type Ad serotype 5 (WT), first generation Ad expressing E. coli beta-galactosidase (AdlacZ), PEGylated AdlacZ (PEGAd), a helper-dependent Ad expressing beta-galactosidase (HDAd), inactivated AdlacZ (UVAd), or phosphate buffered saline (Vehicle) via a jugular cannula. Animals were sacrificed at 0.25, 1, 4, and 14 days. In vitro catalytic activity, protein expression, mRNA, and ALT levels were measured at each timepoint. Six hours after administration of WT, CYP3A2 and 2C11 activity was significantly suppressed (37% and 39%, respectively) as compared to controls. This reduction continued through 14 days with levels falling to 67% (CYP3A2) and 79% (CYP2C11) of controls, P|[le]|0.01. mRNA corresponded to changes in CYP activity. ALT levels of animals treated with this virus were significantly elevated through the 4 day timepoint with respect to levels obtained from saline treated animals (P|[le]|0.05). Use of PEGAd, a vector with a low immunological profile (Croyle et al, Hum Gene Ther, 2002, 13:1887-1900), showed that capsid proteins alone are not responsible for CYP aberrations. CYP3A2 (45%) and 2C11 (42%) was initially suppressed 6 hours after administration (P|[le]|0.05). At 14 days CYP3A2 activity levels remained suppressed (34%, P|[le]|0.05) while CYP2C11 levels recovered. HDAd was administered to investigate the effects of viral gene expression on CYP. Within 6 hours after administration, CYP3A2 (43%) and CYP2C11 (55%) activity was suppressed (P|[le]|0.05). CYP3A2 levels remained suppressed through 14 days (47%, P|[le]|0.01) while CYP2C11 recovered. Preliminary data indicate these alterations occur at the transcriptional level. In contrast, AdlacZ suppressed CYP3A2 and 2C11 activity and mRNA and elevated ALT levels through the 14 day period. UVAd, an inactive control, suppressed CYP3A2 activity by 26% 1 day after administration (P|[le]|0.05) whereas CYP2C11 activity was unaffected throughout the study. These data show that modifications of adenoviral vectors at the molecular and macromolecular level do not eliminate aberrations in CYP following systemic administration and suggest that these changes are not simply the result of the innate immune response. Further studies investigating the role of altered signaling pathways and CYP gene regulatory elements during Ad infection are ongoing.