Shellie M. Callahan

PEGylation of a Vesicular Stomatitis Virus G Pseudotyped Lentivirus Vector Prevents Inactivation in Serum

ABSTRACT One disadvantage of vesicular stomatitis virus G (VSV-G) pseudotyped lentivirus vectors for clinical application is inactivation of the vector by human serum complement. To prevent this, monomethoxypoly(ethylene) glycol was conjugated to a VSV-G-human immunodeficiency virus vector expressing Escherichia coli beta-galactosidase. The modification did not affect transduction efficiency in vitro and protected the vector from inactivation in complement-active human and mouse sera. Blood from mice dosed intravenously with either the unmodified or the PEGylated virus particles was assayed for active vector by a limiting-dilution assay to evaluate transduction efficiency and for p24, an indicator of the total number of virus particles present. PEGylation extended the circulation half-life of active vector by a factor of 5 and reduced the rate of vector inactivation in the serum by a factor of 1,000. Pharmacokinetic profiles for the total number of virus particles present in the circulation were unaffected by PEGylation. Modification of the vector with poly(ethylene) glycol significantly enhanced transduction efficiency in the bone marrow and in the spleen 14 days after systemic administration of the virus. These results, in concert with the pharmacokinetic profiles, indicate that PEGylation does protect the virus from inactivation in the serum and, as a result, improves the transduction efficiency of VSV-G pseudotyped lentivirus vectors in susceptible organs in vivo.

Considerations for use of recombinant adenoviral vectors: Dose effect on hepatic cytochromes P450

Recombinant adenovirus (Ad) serotype 5 is a vector commonly used for gene delivery. Although this vector has a natural tropism for the liver, there is a limited understanding of how Ad administration affects one of the primary hepatic processes, drug metabolism. The effects of systemic administration of a model recombinant adenoviral vector on two hepatic cytochrome P450 (P450) enzymes, CYP3A2 and 2C11, were investigated. Sprague-Dawley rats were treated with one of six vector doses, ranging from 5.7 × 106 to 5.7 × 1012 virus particles (vp)/kg. Hepatic P450 protein expression, catalytic activity, and mRNA levels were measured over 14 days. Ad administration (5.7 × 1010-5.7 × 1012 vp/kg) reduced CYP3A2 over the duration of the study. Six hours after administration of 5.7 × 1012 vp/kg, CYP3A2 activity and mRNA levels were suppressed by 45 and 65%, respectively (P ≤ 0.01). This continued throughout the study with levels dropping to 36 and 45% of controls by 14 days, respectively (P ≤ 0.01). A similar trend was detected for CYP2C11 within this dosing range. Administration of 5.7 × 106, 5.7 × 108, and 5.7 × 109 vp/kg of Ad significantly increased both CYP2C11 protein expression by 86, 71, and 107% and activity 110, 118, and 53%, respectively, above those of animals treated with saline (P ≤ 0.01). These results clearly indicate that a single dose of adenovirus significantly alters key drug metabolizing enzymes for an extended period of time and should be investigated further in the context of the design and implementation of clinical trial protocols.

Impact of transgene expression on drug metabolism following systemic adenoviral vector administration

Systemic administration of a first‐generation adenovirus expressing E. coli beta‐galactosidase (AdlacZ) alters expression and function of two hepatic drug‐metabolizing enzymes, cytochrome P450 (CYP) 3A2 and 2C11, for 14 days. The objective of these studies was to determine how the transgene cassette influences CYP expression and function.

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

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 × 1012 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.

Effect of Sodium Alterations on Hepatic Cytochrome P450 3A2 and 2C11 and Renal Function in Rats

Abstract Numerous dietary supplements are known to modulate cytochrome P450 (CYP)-mediated metabolism and subsequently alter drug toxicity or efficacy in animals and humans. In the present study we investigated the effect of varying amounts of sodium intake on renal function and the metabolic activity of the hepatic CYP3A2 and CYP2C11 isoforms. Rats were maintained on standard rodent chow or a low-salt rice diet. Within each of these groups rats received either a single intraperitoneal injection of furosemide to initiate salt depletion, or saline. Additional groups included salt supplementation of 500 mg/300 g body weight/day and 1.25 g/300 g body weight/day of sodium chloride solution. Rats receiving the low-salt diet, both with and without a concomitant furosemide administration, had a significant reduction in creatinine clearance without changes in serum creatinine. In addition, urine flow rate was markedly reduced in rats maintained on the low-salt diet. Western blot analysis indicated that neither sodium supplementation nor deprivation altered hepatic microsomal CYP3A2 levels; however, hepatic CYP2C11 levels significantly increased in rats receiving the largest sodium supplement. In vitro metabolic activity of CYP3A2 was unchanged as compared with controls. Activity of CYP2C11 was significantly reduced in both rat groups receiving additional sodium supplements. Acute manipulation of daily sodium intake does alter renal function and specific hepatic CYP isoforms and should be considered when using these rat models.

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

Landmark studies describing the effect of microbial infection on the expression and activity of hepatic CYP3A used bacterial lipopolysaccharide as a model antigen. Our efforts to determine whether these findings were translatable to viral infections led us to observations suggesting that engagement of integrin receptors is key in the initiation of processes responsible for changes in hepatic CYP3A4 during infection and inflammation. Studies outlined in this article were designed to evaluate whether engagement of integrins, receptors commonly used by a variety of microbes to enter cellular targets, is vital in the regulation of CYP3A in the presence and absence of virus infection. Mice infected with a recombinant adenovirus (AdlacZ) experienced a 70% reduction in hepatic CYP3A catalytic activity. Infection with a mutant virus with integrin-binding arginine-glycine-aspartic acid (RGD) sequences deleted from the penton base protein of the virus capsid (AdΔRGD) did not alter CYP3A activity. CYP3A mRNA and protein levels in AdlacZ-treated animals were also suppressed, whereas those of mice given AdΔRGD were not significantly different from uninfected control mice. Silencing of the integrin β-subunit reverted adenovirus-mediated CYP3A4 suppression in vitro. Silencing of the α-subunit did not. Suppression of integrin subunits had a profound effect on nuclear receptors pregnane X receptor and constitutive androstane receptor, whereas retinoid X receptor α was largely unaffected. To our knowledge, this is the first time that extracellular receptors, like integrins, have been indicated in the regulation of CYP3A. This finding has several implications owing to the important role of integrins in normal physiologic process and in many disease states.

Evaluation of the HC-04 Cell Line as an In Vitro Model for Mechanistic Assessment of Changes in Hepatic Cytochrome P450 3A During Adenovirus Infection

HC-04 cells were evaluated as an in vitro model for mechanistic study of changes in the function of hepatic CYP3A during virus infection. Similar to in vivo observations, infection with a first generation recombinant adenovirus significantly inhibited CYP3A4 catalytic activity in an isoform-specific manner. Virus (MOI 100) significantly reduced expression of the retinoid X receptor (RXR) by 30% 96 hours after infection. Cytoplasmic concentrations of the pregnane X receptor (PXR) were reduced by 50%, whereas the amount of the constitutive androstane receptor (CAR) in the nuclear fraction doubled with respect to uninfected controls. Hepatocyte nuclear factor 4α (HNF-4α) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) were also reduced by ∼70% during infection. Virus suppressed CYP3A4 activity in the presence of the PXR agonist rifampicin and did not affect CYP3A4 activity in the presence of the CAR agonist CITCO [6-(4-chlorophenyl) imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime], suggesting that virus-induced modification of PXR may be responsible for observed changes in hepatic CYP3A4. The HC-04 cell line is easy to maintain, and CYP3A4 in these cells was responsive to known inducers and suppressors. Dexamethasone (200 μM) and phenobarbital (500 μM) increased activity by 230 and 124%, whereas ketoconazole (10 μM) and lipopolysaccharide (LPS) (10 μg/ml) reduced activity by 90 and 92%, respectively. This suggests that HC-04 cells can be a valuable tool for mechanistic study of drug metabolism during infection and for routine toxicological screening of novel compounds prior to use in the clinic.

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

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.

412. Renal Pathophysiology after Systemic Administration of a Recombinant Adenovirus: Changes in Drug Metabolism Based upon Vector Dose

In addition to its ability to maintain water and electrolyte balance and efficiently remove waste products from the circulation, the kidney actively metabolizes many drugs, hormones and xenobiotics through the cytochrome P450 (CYP) enzyme system. Considering that adenovirus (Ad) significantly alters the expression and function of specific CYP enzymes in the liver, and that slight changes in renal function can adversely affect hepatic CYP activity and expression, we investigated the effect of a model recombinant Ad on the expression of the renal CYP isoforms: 4A1, 4A2, 4F1, and 2E1. CYP4A and 4F convert arachidonic acid to active metabolites responsible for regulation of epithelial transport and vascular function. CYP2E1 oxidizes many small lipophilic compounds and metabolizes carcinogens and cytotoxic agents. Male Sprague-Dawley rats were given one of six doses (5.7 |[times]| 106 - 5.7 |[times]| 1012 virus particles/kilogram (vp/kg)) of Ad expressing E. coli beta-galactosidase or saline (vehicle control) intravenously and sacrificed 0.25, 1, 4, and 14 days after injection. CYP protein expression was measured by Western blot and gene expression by RT-PCR. Serum creatinine levels (SCr) were assessed to profile changes in kidney function. The CYP4A family of proteins were significantly induced within 24 hours of administration of 5.7 |[times]| 1011 vp/kg by 35% and by 47% after 5.7 |[times]| 1012 vp/kg (P|[le]|0.01). A similar increase was detected at the 4 day timepoint. RT-PCR revealed that CYP4A1 gene expression doubled 6 hours after administration of doses above 1010. Gene expression remained induced (70% above control) 1 day after administration of doses, which exceeded 106 vp/kg (P|[le]|0.001). Animals treated with 5.7 |[times]| 1012 vp/kg began to recover at 14 days. However, animals treated with 5.7 |[times]| 1010 and 5.7 |[times]| 1011 vp/kg remained elevated (41% and 67%, P|[le]|0.05). CYP4A2 gene expression was also significantly induced (18%, 24%, and 15%) above control at 1 day in animals treated with 5.7 |[times]| 1010 - 5.7 |[times]| 1012 vp/kg, respectively. This isoform was elevated throughout the duration of the study. CYP4F1 gene and protein expression was unaffected by any of the treatments (P|[ge]|0.05). CYP2E1 gene expression was significantly suppressed by all treatments throughout the study (P|[le]|0.05). In general, suppression peaked within 24 hours (approximately 50% of control) for all doses, but trended towards baseline at 14 days. SCr was significantly decreased at all time points for Ad doses at and below 5.7 |[times]| 1011 vp/kg. SCr was increased by a factor of 3 in animals treated with 5.7 |[times]| 1012 vp/kg 4 days after administration (P|[le]|0.01). This data indicates that systemic Ad treatment can alter renal CYP expression and function up to 14 days after injection. This is of particular importance as Ad genomes were only detected by quantitative real-time PCR in kidneys of animals given the 1011 and 1012 vp/kg doses. This suggests that the aberrations in renal CYPs may not be related to the physical presence of viral particles. This reduction in metabolism should be considered when evaluating the pharmacokinetics of drugs included in clinical protocols involving Ad gene transfer for any application.

148. The Transgene Cassette Is Not Fully Responsible for Alterations of Renal Cytochrome P450 Expression after Systemic Administration of Recombinant Adenovirus

Gene transfer to the kidney has shown promise in treating severe renal disease and prevention of acute renal graft rejection in several animal models. To date, recombinant adenovirus (Ad) is the most efficient vector for gene delivery to the kidney. Although Ad does not infect the kidney efficiently when administered systemically, transgene products secreted from the liver can reach the kidney at therapeutic levels. We have found that systemic administration of recombinant Ad significantly alters the expression of the cytochrome P450 (CYP) enzymes, largely responsible for the clearance of toxicants and the metabolism of xenobiotics and endogenous compounds, in the kidney. In order to determine if the transgene cassette could be partially responsible for this effect, Sprague-Dawley rats received one of four treatments via a surgically implanted catheter: 0.5 ml of phosphate-buffered saline (control) or 5.7|[times]|1012 viral particles/kg of a first generation Ad expressing either E.coli beta-galactosidase (AdlacZ), murine erythropoietin (Epo) or no transgene (Null). Animals were sacrificed 0.25, 1, 4 and 14 days after injection. Renal CYP protein expression and mRNA levels were analyzed by Western blot and RT-PCR. All treatments significantly induced protein levels of the CYP4A family. Following AdlacZ administration, CYP4A increased by 50%, 32% and 64% above control at 0.25, 1 and 4 days, respectively (p<0.01). The vector expressing Epo raised CYP4A protein levels by 42%, 24% and 64% and the Null vector produced elevations of 74%, 25% and 49% above the control at the same timepoints (p<0.01). CYP4A protein expression recovered by 14 days in each group. RT-PCR revealed that mRNA levels of CYP4A2, the primary CYP4A isoform responsible for renal fatty acid metabolism, increased by 16.8% and 19% above control 24 hours after administration of AdlacZ and Epo, respectively (p<0.01). CYP4F1, an isoform responsible for metabolism of fatty acids, prostaglandins and leukotrienes, was unaffected in all treatment groups. In contrast, renal CYP2E1, which metabolizes small lipophilic compounds and carcinogenic agents, was significantly reduced throughout the study. CYP2E1 mRNA levels in animals treated with AdlacZ were 74%, 78%, 69% and 66% of control at 0.25, 1, 4 and 14 days, respectively (p<0.01). Similar reductions were seen with Epo and Null vectors although levels form animals treated with Epo began to recover by 14 days. Real time quantitative PCR revealed that viral genomes (|[sim]|850/100 ng DNA) were initially present in the kidney and declined throughout the study for all treatments. These results suggest that systemic administration of Ad significantly altered the transcription and translation of several key CYP enzymes in the kidney regardless of the transgene expression cassette. This should be considered for accurate drug monitoring when employing recombinant Ad vectors in the clinic. Additional mechanistic studies assessing the effect of expression of Ad early genes and local and systemic eicosanoid biosynthesis on the expression of renal CYP will allow further explanation of these results.