Research paper
Effect of dose on cyclosporine-induced suppression of hepatic cytochrome P450 3A2 and 2C11

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Abstract

Cyclosporine is a potent immunosuppressive drug that undergoes extensive hepatic metabolism catalyzed primarily by the cytochrome P450 (P450) 3A enzyme family. Cyclosporine alters its own metabolism by selective suppression of specific P450 isoforms after chronic therapy in rats. Modulation of hepatic P450 by chronic cyclosporine dosing is associated with increased blood concentrations leading to nephropathy. However, the relationship between cyclosporine dose and hepatic enzyme suppression is not known. The purpose of this study was to examine the effect of escalating doses of cyclosporine on P450 regulation and metabolic activity in the rat. Following 1 week of a low-salt diet, rats were given cyclosporine 5, 15, 30 or 50 mg/kg per day or an equal volume of vehicle for 2 weeks via oral gavage. At the end of the dosing period, livers were removed and hepatic microsomes prepared. Hepatic P450 proteins were measured using Western blot analysis and catalytic activity determined by in vitro testosterone hydroxylation. Cyclosporine dosing suppressed both P450 3A2 and 2C11 protein expression and catalytic activity in a dose-dependent manner. Catalytic activity of two other P450 isoforms, 2A1 and 2B1, were unchanged by cyclosporine administration. Thus, the selective suppression of hepatic microsomal P450 by cyclosporine is not only dependent on the length of therapy, but also the dose administered.

Introduction

Cyclosporine is a potent immunosuppressant of fungal origin that is widely used for the prevention of rejection following allograft transplantation and graft-vs.-host disease prophylaxis after marrow transplantation [1], [2], [3]. Despite its utility, cyclosporine therapy is complicated by significant dose-limiting toxicities to the kidney, liver, and central nervous system. Although these toxicities appear to be dependent on dose and circulating cyclosporine concentrations, prediction of toxicity or efficacy is often difficult.

The primary pathway of cyclosporine elimination is through hepatic metabolism by the cytochrome P450 (P450) enzyme system [4], [5]. Cytochrome P450 is a superfamily of genes whose products represent a divergent group of enzymes related by substrate similarity and amino acid sequence [6]. Currently there are more than 480 different P450 enzymes encoded by more than 70 different gene families in both eukaryotic and prokaryotic species. Despite this large number of enzymes, only a few have been shown to be of importance for the metabolism of therapeutic agents. Even though P450 enzymes are located in numerous tissues, those enzymes located in the liver and small intestine have received the most attention regarding drug absorption and elimination and their subsequent role in therapeutics.

The metabolic pathway of cyclosporine is similar between humans and rodents [7]. Cyclosporine metabolism in the small intestine and liver is primarily by N-demethylation and hydroxylation reactions [8], [9], [10] to three major oxidative metabolites: the 4-N-desmethylated metabolite (AMN4), the monohydroxylated 1-β metabolite (AM1), and the 9-γ-hydroxylated metabolite (AM9). The production of these metabolites is chiefly catalyzed by members of the P450 3A family but also other P450 enzymes are thought to be involved [11]. Male and female juvenile rats constitutively express hepatic P450 3A2, however, upon sexual maturation, only male rats have detectable levels of P450 3A2 protein in liver tissue [12]. Thus, hepatic metabolism of certain substrates in rats is age-dependent.

Cyclosporine is known to alter its own metabolism and toxicity by modulating the activity of selected P450 isoforms [13], [14]. We previously reported that male rats administered cyclosporine for 28 days had a dramatic reduction in hepatic microsomal P450 3A2 and 2C11 protein expression and corresponding metabolic activity as compared with vehicle controls [13]. Female rats given cyclosporine had less of a reduction in kidney function as compared with males, despite female rats having cyclosporine trough blood levels nearly twice those of male counterparts. Furthermore, metabolic activity in female rats was not altered indicating that cyclosporine-mediated alterations in hepatic drug metabolism are gender-dependent and cyclosporine blood levels are not directly related to renal dysfunction in this model.

Inhibition of hepatic P450 is also known to be dependent on the length of dosing [15] and is apparently an indirect process, as in vitro addition of cyclosporine to hepatic microsomal protein does not inhibit microsomal metabolism [16]. Rats were given cyclosporine therapy for up to 28 days followed by 15 days of vehicle only administration. After 2 weeks, cyclosporine treated rats had significant reductions in hepatic P450 3A2 and 2C11 protein levels and in vitro metabolic activity. The greatest reductions were found after 28 days of dosing, with recovery to nearly predosing levels after drug removal and vehicle administration for 15 days. Thus, not only is cyclosporine-dependent suppression directly related to the length of treatment, but the suppression is reversible with the discontinuation of dosing.

The purpose of the study was to determine whether there was a dose-dependent suppression of hepatic P450 protein levels and metabolic activity during chronic cyclosporine administration or whether a threshold dose for suppression exists. We examined the modulation of microsomal oxidative metabolism and protein expression by cyclosporine following 2 weeks of oral dosing using escalating dose of the drug.

Section snippets

Animals

Thirty 10-week-old male Sprague–Dawley rats were purchased from Charles River Breeders (Wilmington, MA) and individually housed in wire bottom cages. Rats were kept in an animal care facility with a 12 h light/dark cycle and controlled temperature and humidity. Following a seven day acclimation period, rats were pair-fed a low sodium diet (17.4% protein, 64.7% carbohydrates, 5.2% fat, 0.05% sodium; Harlan Teklad, Madison, WI) and allowed access to tap water ad libitum. All procedures were

Western blot analysis

In order to monitor the effect of cyclosporine dosing on specific P450 isoforms, immunoblot analysis was performed on hepatic microsomal protein. Microsomes from each rat showed a single immunoreactive band consistent with P450 3A2 whose intensity was inversely related to the dose of cyclosporine administered. Cyclosporine reduced P450 3A2 expression by 27% (P=0.047) in rats given 5 mg/kg per day, by 34% (P=0.015) with 15 mg/kg per day, by 65% (P=0.001) with 30 mg/kg per day, and by 88% (P

Discussion

A clear association between cyclosporine dosing and markers of effect or toxicity does not currently exist in the transplant patient population. The cause of this lack of association is multifactorial, and includes concomitant medications and changes in organ function over time. Even with chronic cyclosporine administration, transplanted patients have an increase in steady-state blood levels which masks the relationship between drug dosing and circulating cyclosporine levels. While this

Acknowledgements

The authors gratefully acknowledge the technical assistance of Minh M. Ha. The study was supported by a research grant from The R.W. Johnson Pharmaceutical Research Institute and by the Donald Seldin, M.D. Young Investigator Grant of the National Kidney Foundation (L.J.B.).

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