Kenneth W. Renton

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.

Alteration of drug biotransformation and elimination during infection and inflammation.

During infection or inflammation, the expression of cytochrome P450 and its dependent biotransformation pathways are modified. This results in a change in the capacity of the liver to handle drugs and in alterations in the production and elimination of endogenous substances throughout the body. The majority of the CYP isoforms are modified at pre-translational steps in protein synthesis, and, in most cases, cytokines are involved as mediators of the response. Recent information suggests that inflammatory responses that are localized to the CNS cause a loss of CYP within the brain. This is accompanied by a parallel down-regulation of CYP in peripheral organs that is mediated by a signaling pathway between the brain and periphery. This review covers the loss that occurs in the major mammalian CYP families in response to infection/inflammation and the mediator pathways that are key to this response.

Inhibition of hepatic microsomal drug metabolism by the calcium channel blockers diltiazem and verapamil

Diltiazem and verapamil were found to be inhibitors of the cytochrome P-450-dependent biotransformation of drugs. Diltiazem and verapamil competitively inhibited the N-demethylation of aminopyrine in hepatic microsomes with Ki values of 100 and 140 microM respectively. Both diltiazem and verapamil were N-demethylated themselves by hepatic microsomes with Km values of 62 and 145 microM respectively. Both drugs also interacted directly with cytochrome P-450 as measured by difference spectra. Diltiazem caused a type I spectral change and verapamil caused a reverse type I spectral change. No metabolic intermediate complexes could be demonstrated for either drug. Inhibition also occurred in vivo as both drugs could prolong pentobarbital-induced sleeping times in mice at doses comparable to those used in man. These results suggest that diltiazem and verapamil may have the potential to cause drug interactions involving inhibition of drug biotransformation.

Regulation of drug metabolism and disposition during inflammation and infection

The expression and activity of cytochrome P450 (CYP) is altered during periods of infectious disease or when an inflammatory response is activated. Most of the major forms of CYP are affected in this manner and this leads to a decrease in the capacity of the liver and other organs to handle drugs, chemicals and some endogenous compounds. The loss in drug metabolism is predominantly an effect resulting from the production of cytokines and the modulation of the transcription factors that control the expression of specific CYP forms. In clinical medicine numerous examples have been reported indicating the occurrence of compromised drug clearance and changes to pharmacokinetics during disease states with an inflammatory component or during infections. For any drug that is metabolised by CYP and has a narrow therapeutic index, there is a significant risk in placing patients in a position where an infection or inflammatory response might lead to aberrant drug handling and an adverse drug 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.

Homogeneous interferon from E. coli depresses hepatic cytochrome P-450 and drug biotransformation

Abstract A highly purified homogeneous human interferon produced from cloned genes depressed the levels of hepatic cytochrome P-450 and related xenobiotic metabolism. Using another cloned human interferon and several impure preparations of human and mouse interferon, it appears that only interferons with antiviral activity in the mouse depress cytochrome P-450 in that species. This is the first direct evidence that interferon decreases hepatic drug biotransformation and likely explains the depression of drug elimination which occurs during viral infections or following the administration of interferon inducers.

Kupffer cell factor mediated depression of hepatic parenchymal cell cytochrome P-450

Following the administration of latex particles (0.46 micron), cytochrome P-450 dependent monooxygenase system was depressed in the livers of mice. These particles were taken up exclusively by Kupffer cells in the liver, and no particles were found in the hepatocytes which contain most of the monooxygenase capacity in that organ. Cytochrome P-450 was also depressed in isolated hepatocytes incubated with phagocytosing Kupffer cells or the cell free filtrate from an incubation mixture of Kupffer cells and latex particles. Kupffer cells and hepatocytes were then incubated in a double-chambered vessel in which the two cell types were separated by a semi-permeable membrane. When latex particles were added to the chamber containing Kupffer cells, a factor was released which crossed the semi-permeable membrane and depressed cytochrome P-450 and benzo[a]pyrene hydroxylase in hepatocytes contained in the other chamber. It is concluded that, during the process of phagocytosis (in vivo or in vitro) by Kupffer cells in the liver, the levels of cytochrome P-450 and related drug biotransformation were depressed in the adjacent parenchymal cells.

Depression of theophylline elimination by erythromycin

The elimination of a single oral dose of theophylline was studied in 12 healthy subjects before and after 250 mg erythromycin every 8 hr for 10 days. Serum theophylline levels were measured by high‐pressure liquid chromatography. Mean theophylline elimination half‐life rose from 4.79 ± 0.43 hr before to 7.53 ± 0.71 hr after erythromycin. Theophylline clearance decreased from a mean of 91.6 ± 27.0 to 54.8 ± 10.0 ml/kg/hr after erythromycin and the mean apparent volumes of distribution were much the same before and after the antibiotic. The excretion of theophylline and its metabolites was studied in the urine of three of the subjects. In each case the amount of 3‐methyixanthine and 1,3‐dimethyluric acid decreased after antibiotic. Adjustments of the theophylline dosage may be necessary for patients who take theophylline and erythromycin concurrently to minimize the risk of theophylline toxicity.

Differential effect of pentoxifylline on lipopolysaccharide-induced downregulation of cytochrome p450☆

It is now established that inflammatory stimuli such as lipopolysaccharides (LPS) and polyinosinic acid:polycytidylic (polyIC) suppress hepatic expression of cytochrome P450 (P450) genes in rat liver. Previous studies have suggested that LPS- or polyIC-induced downregulation of P450 was due to endogenously released inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1, interleukin-6, and interferons (IFNs). To improve our understanding of the role of inflammatory cytokines in mediating P450 depression, we investigated the possibility of preventing P450 downregulation with pentoxifylline. Pentoxifylline has been shown to inhibit LPS-induced TNF-alpha production by suppression of TNF-alpha gene expression. The present study shows that in uninduced male rats pentoxifylline selectively prevents the downregulation of microsomal P4501A2 and P4502B caused by LPS. No protective effect of pentoxifylline on the downregulation of P4502E1 and P4503A1/2 was observed. PolyIC-induced downregulation of P4501A2, P4502B, P4502E1, and P4503A1/2 was not affected by pentoxifylline. These results suggest that the LPS-induced downregulation of P4501A2 and P4502B is mediated to a large extent by TNF-alpha. Other cytokines might be involved in the suppression of P4502E1 and P4503A1/2. The fact that polyIC-induced downregulation is not protected by pentoxifylline is further evidence that this agent acts via a selective induction of IFNs.

Cytochrome P4501A1 and cytochrome P4501A2 are downregulated at both transcriptional and post-transcriptional levels by conditions resulting in interferon-α/β induction

Abstract The interferon mediated downregulation of constitutive and inducible cytochrome P450 enzymes occurs through a pretranslational mechanism which depresses the mRNA encoding cytochrome P450s. We measured the transcription rates of CYP1A genes and the turnover of CYP1A mRNA in rats treated with the interferon-α/β inducer polyinosinic acid-polycytidylic acid. The rate of transcription of CYP1A1 and CYP1A2 genes was significantly decreased in hepatic nuclei isolated from male rats treated with polyinosinic acid-polycytidylic acid (10 mg kg ). In addition the rate of degradation of hepatic CYP1A1 and CYP1A2 mRNA was examined following the inhibition of de novo transcription by actinomycin D (l mg kg ). Messenger RNA levels were analysed by Northern and slot blotting with a 1.2 kb murine CYP1A1 cDNA probe. Interferon significantly augmented the rate of loss of CYP1A1 and CYP1A2 mRNAs suggesting that post-transcriptional degradation of mRNA contributes to the pre-translational events that cause cytochrome P450 downregulation. These results support the involvement of both transcriptional and post-transcriptional mechanisms in the loss of cytochrome P450s mediated by interferon inducers.