Micheline Piquette-Miller

Regulation of Drug-Metabolizing Enzymes and Transporters in Infection, Inflammation, and Cancer

This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 07 meeting in Washington, DC. The presentations discussed the phenomenology, clinical consequences, and underlying mechanisms of cytochrome P450 and drug transporter regulation by inflammatory and infectious stimuli. Although considerable insights into the links between inflammatory mediators and altered hepatic drug clearance pathways have been gained from previous studies with acute inflammatory stimuli, this symposium highlighted recent advances in understanding how these processes operate in other organs and chronic inflammatory states relevant to human diseases. The development of mouse models of live bacterial infection provides excellent opportunities to explore the impact of infection on drug metabolism beyond the well characterized effects of bacterial endotoxin. Altered levels of cytochromes P450 and especially drug transporters due to inflammation in brain, intestine, and placenta have significant implications for the use of many drugs in diverse clinical settings. The consequences of inflammatory cytokine production by tumors for drug safety and efficacy in cancer patients were outlined. Repression of drug clearance pathways by tumor-derived cytokines may result in extreme toxicity to chemotherapy, compromising treatment of many cancers. It is fitting that, in honoring the career contributions and achievements of Dr. Kenneth W. Renton, this symposium reinforced the clinical relevance of this field.

Decreased Expression and Activity of P-GIycoprotein in Rat Liver During Acute Inflammation

AbstractPurpose. Drug disposition is often altered in inflammatory disease. Although the influence of inflammation on hepatic drug metabolism and protein binding has been well studied, its impact on drug transport has largely been overlooked. The multidrug resistance (MDR) gene product, P-glycoprotein (P-gp) is involved in the active secretion of a large variety of drugs. Our goal was to ascertain the influence of acute inflammation (AI) on the expression and functional activity of P-gp. Methods. AI was induced in rats through turpentine or lipopolysaccharide (LPS) administration. Expression of P-gp in liver was detected at the level of protein on Western blots using the monoclonal antibody C-219 and at the level of mRNA using an RNase protection assay. P-gp mediated transport activity was assessed by measuring the verapamil-inhibitable efflux of rhodamine 123 (R123) in freshly isolated hepatocytes. Results. Turpentine-induced AI significantly decreased the hepatic protein expression of P-gp isoforms by 50−70% and caused a significant 45−65% reduction in the P-gp mediated efflux of R123. Diminished mRNA levels of all three MDR isoforms were seen. LPS-induced AI similarly resulted in significantly reduced levels and activity of P-gp in liver. Although differences in the constitutive levels of P-gp were seen between male and female rats, the influence of AI on P-gp expression and activity was not gender specific. Conclusions. Experimentally-induced inflammation decreases the in vivo expression and activity of P-gp in liver. This is the first evidence that expression of P-gp is modulated in response to experimentally-induced inflammation.

The involvement of the pregnane X receptor in hepatic gene regulation during inflammation in mice

Inflammation and proinflammatory cytokines suppress the expression of several hepatic transporters and metabolic enzymes, often resulting in cholestatic liver disease. However, mechanism(s) of this down-regulation have not been fully elucidated. As the pregnane X receptor (PXR) is involved in inducing many of these hepatic proteins, it is possible that PXR is also involved in their down-regulation during inflammation. Thus, we compared the effect of inflammation on hepatic gene regulation in wild-type (PXR+/+) versus PXR-null (PXR-/-) mice. Treatment of PXR+/+ but not PXR-/- mice with the PXR activators 5-pregnen-3β-ol-20-one-16α-carbonitrile (PCN) or 17β-hydroxy-11β-[4-dimethylamino phenyl]-17α-[1-propynyl] estra-4,9-dien-3-one (RU486) resulted in increased mRNA levels of bsep, mdr1a, mrp2, mrp3, oatp2, and cyp3a11, indicating involvement of PXR in their regulation. Significantly lower mRNA levels of bsep, mdr2, mrp2, mrp3, ntcp, oatp2, and cyp3a11 were found in endotoxin-treated PXR+/+ mice. In endotoxin-treated PXR-/- mice, the extent of mrp2 suppression was significantly diminished. Changes in MRP2 expression were supported by Western blot analysis. Although interleukin (IL)-6 imposed significant decreases in the expression of bsep, mrp2, and cyp3a11 in PXR+/+ mice, this was not observed in PXR-/- mice. Of note, significantly lower levels of PXR mRNA and protein were detected in endotoxin- and IL-6-treated PXR+/+ mice. In addition, endotoxin and IL-6 were also able to suppress PCN-mediated induction of bsep, mrp2, cyp3a11, and PXR. Taken together, our results suggest that PXR plays a role in the down-regulation of several hepatic proteins during inflammation.

Regulation of the hepatic multidrug resistance gene expression by endotoxin and inflammatory cytokines in mice

P-glycoprotein (PGP), an ATP-dependent membrane transporter is found in epithelial tissues of the liver, kidneys, intestine and blood-brain barrier. In tumor cells, PGP is often overexpressed and confers multidrug resistance toward cancer chemotherapeutics. It has been previously shown in rats that induction of an inflammatory response evokes a decrease in hepatic expression of PGP. In order to identify the inflammatory mediators involved in this phenomenon, we examined the influence of experimentally induced inflammation and pro-inflammatory cytokines (interleukin (IL)-6, IL-1beta and tumor necrosis factor (TNF)-alpha) on the hepatic expression of PGP in mice. A significant reduction in the hepatic expression of mdr1a, mdr1b, mdr2 and spgp genes were seen in endotoxin (lipopolysaccharide (LPS)) and turpentine-treated mice. Similarly, IL-6-treated mice displayed a 70% reduction in protein expression and a 40-70% reduction in the mRNA levels of all PGP mdr isoforms. Administration IL-1beta caused an increase in both mdr1b mRNA and protein expression, however, mRNA levels of mdr1a, mdr2 and spgp were significantly reduced. Administration of TNF-alpha also caused increases in mdr1b mRNA. These findings indicate that IL-6 plays a principal role in the downregulation of PGP that is observed in the livers of mice during an inflammatory response.

Downregulation of mdr1a expression in the brain and liver during CNS inflammation alters the in vivo disposition of digoxin

Inflammation is a pathophysiological event that has relevance for altered drug disposition in humans. Two functions of P‐glycoprotein (P‐gp) are hepatic drug elimination and prevention of drug entry into the central nervous system (CNS). Our objective was to investigate if localized CNS inflammation induced by Escherichia coli lipopolysaccharide (LPS) would modify mdr1a/P‐gp expression and function in the brain and liver. Our major finding was that the CNS inflammation in male rats produced a loss in the expression of mdr1a mRNA in the brain and liver that was maximal 6 h after intracranial ventricle (i.c.v.) administration of LPS. When 3H‐digoxin was used at discrete time points, as a probe for P‐gp function in vivo, an increase in brain and liver 3H‐radioactivity and plasma level of parent digoxin was produced 6 and 24 h following LPS treatment compared to the saline controls. Digoxin disposition was similarly altered in mdr1a+/+ mice but not in mdr1a−/− mice 24 h after administering LPS i.c.v. In male rats, the biliary elimination of parent digoxin was reduced at 24 h (60%) and 48 h (40%) after LPS treatment and was blocked by the P‐gp substrate cyclosporin A. An observed loss in CYP3A1/2 protein and organic anion transporting polypeptide 2 mRNA in the liver may make a minor contribution to digoxin elimination in male rats after LPS treatment. Conditions which impose inflammation in the CNS produce dynamic changes in mdr1a/P‐gp expression/function that may alter hepatic drug elimination and the movement of drugs between the brain and the periphery. The use of experimental models of brain inflammation may provide novel insight into the regulation of P‐gp function in that organ.

Hepatoprotective role of PXR activation and MRP3 in cholic acid‐induced cholestasis

Activation of the pregnane X receptor (PXR) has been shown to protect against cholestatic hepatotoxicity. As PXR alters the expression of numerous hepatic bile acid transporters, we sought to delineate their potential role in hepatoprotection.

Functional and Molecular Characteristics of Na+-dependent Nucleoside Transporters

Nucleoside transporters play a critical role in the absorption, disposition, and targeting of therapeutically used nucleosides and nucleoside analogs. This review is focused on the Na+-dependent, concentrative nucleoside transporters which are found in a variety of cells including renal, intestinal and hepatic epithelia. Five major Na+-dependent nucleoside transporter subtypes have been characterized in isolated tissue preparations: Nl is purine selective; N2 is pyrimidine selective and N3−N5 exhibit variable selectivity for both purine and pyrimidine nucleosides. The recent cloning of Nl and N2 nucleoside transporters has provided the first information on the molecular function and structure of concentrative nucleoside transporters. In this manuscript we review the characteristics of the various subtypes of nucleoside transporters and the molecular structure, functional properties, and tissue distribution of the cloned Na+-dependent nucleoside transporters. In addition, the interactions of nucleosides and nucleoside analogs with the cloned transporters in mammalian and amphibian expression systems are presented. Mammalian expression systems may be particularly useful during drug development in screening potential compounds for improved bioavailability and tissue specific targeting. Finally, we present our view of future areas of study in the field of nucleoside transporters.

Decreased expression of P-glycoprotein in interleukin-1β and interleukin-6 treated rat hepatocytes

Abstract.Objective and Design: As acute inflammation is known to cause a reduction in hepatic P-Glycoprotein (PGP) expression and activity in rats, we tested the hypothesis that the pro-inflammatory cytokines interleukin (IL-)1β and IL-6 also mediate reductions in PGP.¶Methods: Hepatocytes were incubated with 0-50 ng/ml of cytokine for 24-72 h. PGP/mdr expression was examined by immunodetection and quantitative RT-PCR analysis and PGP efflux activity was assayed.¶Results: PGP protein was significantly reduced in cells treated for 3 days with IL-1β and 24 h with IL-6 (p < 0.05), maximal effects occurring at 5 ng/ml for each cytokine. PGP activity was reduced in both IL-1β and IL-6 treated cells (p < 0.05). mdr1 mRNA was decreased in cells treated with IL-6, but not IL-1β. spgp and mdr2 were not affected.¶Conclusions: Our data indicate that IL-6 and IL-1β have suppressive effects on the expression and activity of PGP in cultured hepatocytes, likely occurring through distinct mechanisms. These cytokines may have a potential role in PGP regulation during inflammatory responses.

Synthesis and Physicochemical and Dynamic Mechanical Properties of a Water-Soluble Chitosan Derivative as a Biomaterial

The physicochemical and rheological properties of a water-soluble chitosan (WSC) derivative were characterized in order to facilitate its use as a novel material for biomedical applications. The WSC was prepared by conjugating glycidyltrimethylammonium chloride (GTMAC) onto chitosan chains. Varying the molar ratio of GTMAC to chitosan from 3:1 to 6:1 produced WSCs with a degree of substitution (DS) that ranged from 56% to 74%. The WSC with the highest DS was soluble in water up to concentrations of 25 g/dL at room temperature. An increase in the polymer concentration gradually increased both the pH and conductivity of the WSC solutions. The rheological properties of the WSC solutions were found to be dependent on the salt and polymer concentrations as well as the DS value. In the absence of salt, the rheological behavior of the WSC was found to be typical of that for a polyelectrolyte in the dilute solution regime. However, the addition of salt decreased the viscosity of the polymer solution due to the reduction of electrostatic repulsions by the positively charged trimethylated ammonium groups of the WSC. In the concentrated regime, the viscosity of the WSCs was found to follow a power-law expression. The lowest DS WSC had the more favorable viscoelastic properties that were attributed to its high molecular weight, as confirmed by the stress relaxation spectra and intrinsic viscosity measurements. The effect of DS on the degree of interaction between WSC and the lipid egg phosphatidylcholine was investigated by FTIR analysis. Overall, the lower DS WSC had enhanced rheological properties and was capable of engaging in stronger intermolecular physical interactions.

Cellular localization and functional expression of P‐glycoprotein in rat astrocyte cultures

We investigated the cellular/subcellular localization and functional expression of P‐glycoprotein, an ATP‐dependent membrane‐associated efflux transporter, in astrocytes, a brain parenchyma compartment that is poorly characterized for the expression of membrane drug transporters. Analyses were carried out on primary cultures of astrocytes isolated from the cerebral cortex of neonatal Wistar rats and CTX TNA2, an immortalized rat astrocyte cell line. Both cell cultures display morphological features typical of type I astrocytes. RT‐PCR analysis revealed mdr1a and mdr1b mRNA in primary cultures of astrocytes and in CTX TNA2 cells. Western blot analysis using the P‐glycoprotein monoclonal C219 antibody detected a single band of appropriate size in both cell systems. Immunocytochemical analysis using the monoclonal antibodies C219 and MRK16 labeled P‐glycoprotein along the plasma membrane, caveolae, coated vesicles and nuclear envelope. Immunoprecipitation studies using the caveolin‐1 polyclonal H‐97 antibody demonstrated that P‐glycoprotein is physically associated with caveolin‐1 in both cell culture systems. The accumulation of [3H]digoxin (an established P‐glycoprotein substrate) by the astrocyte cultures was significantly enhanced in the presence of standard P‐glycoprotein inhibitors and an ATP depleting agent. These results demonstrate the cellular/subcellular location and functional expression of P‐glycoprotein in rat astrocytes and suggest that this glial compartment may play an important role in the regulation of drug transport in the CNS.