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 integrinß-subunit reverted adenovirus-mediated CYP3A4 suppression in vitro. Silencing of theα-subunit did not. Suppression of integrin subunits had a profound effect on nuclear receptors pregnane X receptor and constitutive androstane receptor, whereas retinoid X receptorαwas 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.

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 x 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

Controlled inactivation of recombinant viruses with vitamin B2.

Inactivated viruses are important tools for vaccine development and gene transfer. 8-Methoxypsoralen (8-MOP) and long-wavelength ultraviolet irradiation (LWUVI) inactivates many viruses. Toxicity limits its use in animals and humans. Toxicological and photosensitizing properties of riboflavin make it suitable for virus inactivation in preparations for biological use. Viruses expressing beta-galactosidase were mixed with either 8-MOP (1.5mM) or riboflavin (50 microM) and exposed to LWUVI (365 nm) for 2 h. Virus activity was determined by limiting dilution. The half-life of the adenovirus preparation treated with 8-MOP was 8.28 ns(-1) and 36.5 ns(-1) after treatment with riboflavin. Despite the difference in half-life, both preparations were completely inactivated within 45 min. In contrast, the half-lives for adeno-associated virus (AAV) preparations were similar (63 ns(-1) 8-MOP vs. 67 ns(-1) riboflavin). Each AAV preparation was fully inactivated within 90 min. The half-life of lentivirus was 193.4 ns(-1) after treatment with 8-MOP and 208 ns(-1) after exposure to riboflavin. Virus treated with riboflavin was inactivated within 20 min. Virus exposed to 8-MOP was inactivated in 90 min. DNA and RNA viruses can be inactivated by riboflavin and LWUVI and used in physiological systems sensitive to other photochemicals.

Renal pathophysiology after systemic administration of recombinant adenovirus: changes in renal cytochromes P450 based on vector dose.

Recombinant adenovirus (Ad) significantly alters hepatic cytochrome P450 (CYP). Because changes in renal function can alter hepatic CYP, the effect of Ad on renal CYPs 4A1, 4A2, 4F1, and 2E1 was evaluated. Male Sprague-Dawley rats were given one of six intravenous doses (5.7x10(6)-5.7x10(12) viral particles/kg [VP/kg]) of Ad expressing beta-galactosidase or saline. CYP protein, activity, gene expression, and serum creatinine (SCr) were evaluated 0.25, 1, 4, and 14 days later. Doses of 5.7x10(11) and 5.7x10(12) VP/kg increased CYP4A protein within 24 hr by 35 and 48%, respectively (p<0.05). A similar trend was observed on day 4. CYP4A1 mRNA doubled 6 hr after doses of 5.7x10(10)-10(12) VP/kg (p<0.01). Similar effects were observed 1 day after each dose tested. CYP4A2 gene expression was 20% above control 1 day after treatment with 5.7x10(10)-10(12) VP/kg and remained high through day 14. CYP4F1 expression was unaffected by all doses (p=0.08). CYP2E1 activity and gene expression were significantly suppressed 24 hr after administration of all doses and began to normalize by day 14 (p<0.01). SCr was significantly reduced (approximately 50%) throughout the study for doses at and below 5.7x10(11) VP/kg. SCr was increased by a factor of 3 by 5.7x10(12) VP/kg and glomerular filtration was significantly reduced (p<0.01). This suggests that changes in renal CYP and corresponding arachidonic acid metabolites may play a role in the documented toxicity associated with the systemic administration of recombinant Ad.

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

BACKGROUND: 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. METHODS: Sprague-Dawley rats were given 5.7 x 10(12) viral particles (vp)/kg of either: AdlacZ, Ad expressing murine erythropoietin (Epo), Ad without a transgene (Null), or phosphate-buffered saline (Vehicle). Hepatic CYP protein expression, activity, mRNA and alanine aminotransferase (ALT) levels were analyzed 0.25, 1, 4, and 14 days following a single intravenous injection. RESULTS: Administration of Epo did not alter CYP3A2 activity, but induced RNA levels by a factor of 2 at 4 and 14 days (P< or =0.01). This vector suppressed CYP2C11 activity levels by 45% at 1 day (P< or =0.05) and RNA levels throughout the study period (P< or =0.05). The Null vector suppressed CYP3A2 activity by 36, 63, 34, and 45% at 0.25, 1, 4 and 14 days, respectively (P< or =0.05). CYP2C11 activity was suppressed 1 day after administration (41%) and RNA levels were suppressed at 6 h (53%) and 1 day (36%, P< or =0.05). In contrast, AdlacZ suppressed both CYP3A2 and 2C11 at all time points. CONCLUSIONS: The immunogenic and biological nature of the transgene cassette can influence changes in CYP3A2, but not the 2C11 isoform. The shift in transcription and translation of protein for maintenance of physiologic homeostasis to production of viral proteins and transgene product and their associated toxicity during viral infection may explain our observations.

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 x 10(6) to 5.7 x 10(12) virus particles (vp)/kg. Hepatic P450 protein expression, catalytic activity, and mRNA levels were measured over 14 days. Ad administration (5.7 x 10(10)-5.7 x 10(12) vp/kg) reduced CYP3A2 over the duration of the study. Six hours after administration of 5.7 x 10(12) vp/kg, CYP3A2 activity and mRNA levels were suppressed by 45 and 65%, respectively (P < or = 0.01). This continued throughout the study with levels dropping to 36 and 45% of controls by 14 days, respectively (P < or = 0.01). A similar trend was detected for CYP2C11 within this dosing range. Administration of 5.7 x 10(6), 5.7 x 10(8), and 5.7 x 10(9) 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 < or = 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.

Cyclosporine and bromocriptine-induced suppressions of CYP3A1/2 and CYP2C11 are not mediated by prolactin.

The purpose of this study was to determine if the suppression of hepatic CYP3A1/2 (cytochrome P450 3A1/2) and CYP2C11 (cytochrome P450 2C11) by cyclosporine is mediated by prolactin. Male intact rats were given subcutaneous doses of either 15 mg/kg/day of cyclosporine or 1 ml/kg/day of cyclosporine vehicle concomitantly with one of the following: 500 mg/kg prolactin, 1 ml/kg prolactin vehicle, 4 mg/kg bromocriptine, or 1 ml/kg bromocriptine vehicle for 14 days. Protein expressions were measured using Western blot analysis and activities were measured using an in vitro testosterone hydroxylation assay. The administration of prolactin did not significantly alter CYP3A1/2 protein expression. Hypoprolactinemia, produced by bromocriptine, caused a significant suppression of CYP3A1/2 activity levels when bromocriptine was administered alone and in combination with cyclosporine (P<0.001, P<0.05; respectively). However, the cause of the suppression by bromocriptine was likely not the result of lowering prolactin levels. Bromocriptine administration also lowered CYP2C11 protein expression and activity, while prolactin administration had virtually no effect on CYP2C11. Chronic cyclosporine administration caused a 140% increase in prolactin area under the curve (AUC) (P<0.001). Bromocriptine caused a significant decline in endogenous prolactin secretion, however, concurrent cyclosporine administration did not recover these levels. Overall, while both cyclosporine and bromocriptine, separately, can significantly alter the fate of hepatic P450 enzymes, the suppression is likely not due to an alteration in prolactin levels.

PEGylation of a vesicular stomatitis virus G pseudotyped lentivirus vector prevents inactivation in serum.

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.

Effect of high-density lipoprotein associated cyclosporine on hepatic metabolism and renal function.

Cyclosporine (CSA), in both humans and animals, is associated with plasma lipoproteins. It has been demonstrated that CSA-lipoprotein association is partly responsible for the distribution and toxicity related to CSA use. Altered plasma lipoprotein profiles are often seen in transplantation recipients undergoing CSA treatment. In the present study, daily 0.1 mg/kg intravenous injections of either high-density lipoprotein-associated CSA (HDL-CSA), plasma-associated CSA (Plasma-CSA), or CSA in Cremophor (CSA) were administered to adult male rats for 14 days. Vehicle controls included daily administrations of 0.5 ml/kg of Cremophor or saline. Serum creatinine levels, a marker of renal function, increased in rats administered Plasma-CSA as compared with control rats treated with CSA. CYP3A and CYP2C11 protein expression was suppressed by 27% and 39%, respectively, in the HDL-CSA treatment group and by 38% and 40% in the Plasma-CSA treated group as compared with CSA controls. In addition, 6beta-hydroxytestosterone, a marker of CYP3A activity, was reduced by 33% and 34% in the HDL-CSA and the Plasma-CSA treatment groups, respectively, as compared with the CSA control group. CYP2C11 activity was measured by the in vitro formation of 2alpha-hydroxytestosterone. Activity levels in rats treated with HDL-CSA and Plasma-CSA were slightly induced as compared to CSA controls, however these differences were not found to be statistically significant. In summary, Plasma-CSA treatment resulted in renal dysfunction and suppressed CYP3A and CPY2C11 protein expression. These results demonstratethat intravenous lipoprotein-associated CSA alters the metabolism of CSA in the rat differently than CSA alone.

Effect of sodium alterations on hepatic cytochrome P450 3A2 and 2C11 and renal function in rats.

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.

Suppression of hepatic CYP3A1/2 and CYP2C11 by cyclosporine is not mediated by altering growth hormone levels.

Cyclosporine (CsA) suppresses drug metabolism by decreasing cytochrome P450 (P450) enzyme levels in rat liver. Growth hormone (GH) is known to pretranslationally regulate P450 expression. Thus, the suppression of P450 by CsA may involve GH as an intermediate. To address this question, we examined the effects of administering exogenous GH via twice daily subcutaneous injections and in conjunction with chronic subcutaneous CsA administration for 14 days on hepatic P450 expression. CsA alone decreased CYP3A1/2 and CYP2C11 significantly, in a manner similar to that previously found. When administered in the absence of CsA, GH also suppressed CYP3A1/2 and CYP2C11 protein levels as compared with GH vehicle. In the presence of CsA, GH did not cause further suppression of either CYP3A1/2 or CYP2C11 expression when compared with CsA treatment with GH vehicle. Testosterone in vitro catalytic assays confirmed that CsA and GH separately cause significant decreases in activity levels. Also, the concomitant administration of GH and CsA caused lowered production of 16alpha-, 2alpha-, 6beta-, and 2beta-hydroxytestosterone as compared with the administration of GH with CsA vehicle and as compared with the administration of GH vehicle with CsA. This study shows that GH is a dominating factor over CsA in determining hepatic P450 expression and activity. In addition, CsA does not seem to alter GH levels as a mediating event in suppressing P450 expression and activity. Since CsA given in combination with GH further suppressed P450 activity as compared with CsA given in combination with vehicle, this suggests that changes in hormonal status are likely to be one of the many factors that is responsible for the lack of a clear association between cyclosporine dosing and markers of toxicity.