Molecular and macromolecular alterations of recombinant adenoviral vectors do not resolve changes in hepatic drug metabolism during infection

Abstract

In this report we test the hypothesis that long-term virus-induced alterations in CYP occur from changes initiated by the virus that may not be related to the immune response. Enzyme activity, protein expression and mRNA of CYP3A2, a correlate of human CYP3A4, and CYP2C11, responsive to inflammatory mediators, were assessed 0.25, 1, 4, and 14 days after administration of several different recombinant adenoviruses at a dose of 5.7 × 10 12 virus particles (vp)/kg to male Sprague Dawley rats. Wild type adenovirus, containing all viral genes, suppressed CYP3A2 and 2C11 activity by 37% and 39%, respectively within six hours. Levels fell to 67% (CYP3A2) and 79% (CYP2C11) of control by 14 days (p ≤ 0.01). Helper-dependent adenovirus, with all viral genes removed, suppressed CYP3A2 (43%) and CYP2C11 (55%) within six hours. CYP3A2 remained significantly suppressed (47%, 14 days, p ≤ 0.01) while CYP2C11 returned to baseline at this time. CYP3A2 and 2C11 were reduced by 45 and 42% respectively 6 hours after treatment with PEGylated adenovirus, which has a low immunological profile (p ≤ 0.05). CYP3A2 remained suppressed (34%, p ≤ 0.05) for 14 days while CYP2C11 recovered. Inactivated virus suppressed CYP3A2 activity by 25-50% for 14 days (p ≤ 0.05). CYP2C11 was affected similar manner but recovered by day 14. Microarray and in vitro studies suggest that changes in cellular signaling pathways initiated early in virus infection contribute to changes in CYP.

Integrin Receptors Play a Key Role in the Regulation of Hepatic CYP3A

“…Although the primary paradigm of the field attributed this effect to the production of interferons, cytokines, and chemokines (Zanger and Schwab, 2013), we found that the expression and function of hepatic CYP3A2 in the rat is suppressed for 14 days after a single dose of a recombinant adenovirus, long after these inflammatory mediators dissipate (Callahan et al, 2005). Additional studies revealed that reducing the immunogenicity of the virus by chemical and physical means did not mitigate this effect (Callahan et al, 2008a). These findings (coupled with a clinical report detailing minimal changes in hepatic CYP3A in patients given exogenous cytokines in the absence of infection; Reiss and Piscitelli, 1998) suggest that the presence of the microbial pathogen itself plays a role in the process that alters CYP3A during infection.…”

“…hepatocytes (Callahan et al, 2008a), making this cell line useful for further mechanistic studies to determine how integrins regulate CYP3A4 in humans.…”

“…On a transcriptional level, hepatic CYP3A is constitutively expressed in the liver, but several nuclear receptors, including PXR, CAR, and RXR, are primarily responsible for regulating CYP3A gene expression (Croyle, 2009). However, it remains to be known which is predominantly influenced by virus infection and is ultimately responsible for our observed changes in CYP3A (Callahan et al, 2008a;Wonganan et al, 2014). In an effort to determine how integrin engagement changes each of these key regulators of CYP3A in vivo, gene and protein expression was determined 24 and 48 hours after infection with each virus (Fig.…”

“…During some of the initial studies conducted in our laboratory in which we characterized CYP3A expression and function in the rat (Callahan et al, 2005(Callahan et al, , 2006(Callahan et al, , 2008a, samples of liver tissue were taken and processed for assessment of changes in genes associated with different signal transduction patterns. Samples were taken 4 days after administration of four different live and inactivated recombinant adenovirus vectors: AdlacZ (a first-generation E1/E3 deleted adenovirus serotype 5 virus expressing the Escherichia coli b-galactosidase transgene under the control of a cytomegalovirus promoter), PEGAd (the AdlacZ virus modified by covalent attachment of PEG to the virus capsid, which reduces the immune response to the virus; Croyle et al, 2001Croyle et al, , 2002, HDAd (a third-generation adenovirus in which all elements of the viral genome except the inverted terminal repeats and the packaging signal [C] are removed and which also contains the constitutive androstane receptorgalactosidase transgene; Croyle et al, 2005), and UVAd (the AdlacZ vector inactivated by exposure to UV light; Callahan et al, 2008b).…”

“…Each of these pathways has been documented to alter the expression and function of hepatic CYP3A in some capacity (Ding and Staudinger, 2005;Wang et al, 2008;Zangar et al, 2008;Pondugula et al, 2009;Kacevska et al, 2013). Further evaluation of these data with that from a pilot study in which CYP3A activity was suppressed in primary hepatocytes treated with an arginine-glycine-aspartic acid (RGD) peptide known to engage integrins in the absence of virus infection (Callahan et al, 2008a) solidified our hypothesis that engagement of integrin receptors by the virus at the start of infection may be sufficient to initiate processes that suppress hepatic CYP3A.…”