Determination of the Enzyme(s) Involved in the Metabolism of Amiodarone in Liver and Intestine of Rat: The Contribution of Cytochrome P450 3a Isoforms

Abstract

ABSTRACT:In humans, cytochrome P450 3A (CYP3A4) is a major enzyme involved in the metabolism of amiodarone (AM) to its major metabolite, desethylamiodarone (DEA). In rat, a commonly used animal model, metabolism of AM has not been well studied. To determine whether DEA is formed by CYP3A isoenzymes in the rat, microsomal protein was harvested from liver and intestine of male Sprague-Dawley rats. The metabolism of AM in each tissue was assessed utilizing chemical and immunological inhibitors. Ketoconazole, a presumed inhibitor of CYP3A1/2, significantly inhibited formation of DEA by hepatic and intestinal microsomes. However, based on the DEA formation kinetics in both microsomal preparations, it appeared that more than one cytochrome P450 enzyme was involved in the process. Coincubation of AM with microsomes and anti-CYP3A2 confirmed the role of CYP3A2 in the metabolism of AM in liver. DEA was also formed by rat recombinant CYP1A1 and CYP3A1, and was inhibited by ketoconazole; hence the participation of these enzymes in the intestinal DEA formation is likely. However, anti-CYP2B1/2 or -CYP1A2 antibodies had no effect on DEA formation. In rats given oral or intravenous AM, oral ketoconazole caused significant increases in area under the concentration versus time curve (AUC) of oral and i.v. treated rats and greater than 50% decreases in the total body clearance and V dss of i.v. treated rats. Although low to undetectable concentrations of DEA were a limitation for determination of AUC of DEA in vivo, it was confirmed that ketoconazole could cause a significant increase in AM concentrations in rat.Amiodarone (AM) is an iodinated class III antiarrhythmic benzofuran derivative with extensive clinical usage (Trivier et al., 1993;Soyama et al., 2002) in the treatment of life-threatening ventricular and supraventricular arrhythmias (Naccarelli et al., 2000). AM undergoes extensive hepatic biotransformation (Trivier et al., 1993;Soyama et al., 2002). Among the five pathways involved in the metabolism of AM (N-deethylation, hydroxylation, O-dealkylation, deiodination, and glucuronidation), N-dealkylation is most important in humans (Trivier et al., 1993;Soyama et al., 2002). The dealkylated metabolite, desethylamiodarone (DEA) shares some of the pharmacological and toxicological properties of AM. For instance, some of the electrocardiographic changes observed after long-term therapy with AM might be related to DEA (Kharidia and Eddington, 1996;Kodama et al., 1999). In addition, both AM and DEA inhibit the intracellular conversion of thyroxine to triiodothyronine. This inhibition may be related to some cardiotoxic effects of AM such as bradycardia and reduced myocardial oxygen consumption, and may explain the hypothyroid-like condition observed after chronic administration of AM (Hudig et al., 1994;Kodama et al., 1999).AM possesses a very large pharmacokinetic volume of distribution (V d ) and extensive tissue distribution, and, in turn, a long terminal half-life (t 1/2 ) in rat and human plasma (Pollak et al., 2000;Shaye...

The metabolism of amiodarone by various CYP isoenzymes of human and rat, and the inhibitory influence of ketoconazole

“…The fraction of the dose of AM converted to DEA was recently estimated to be up to 64% in rat (8). Earlier reports have demonstrated the involvement of CYP3A4, 1A1, 2D6 and 2C8 in DEA formation in human whereas CYP3A1 and 1A1 were found to be involved in DEA formation in rats (9)(10)(11). Similar to the parent drug, DEA possesses antiarrhythmic activity of its own.…”

“…An HPLC method was used for analysis of AM and DEA (11,28). The assay had a validated lower limit of quantitation of 35 ng/ml for both AM and DEA based on 100 µL of rat plasma (28).…”

“…In rat this interaction is characterized by a 24% increase in overall bioavailability of orally administered AM (11). On the other hand, in humans the interaction of AM with azole antifungal agents is well recognized and could lead to serious clinical side effects such as Torsades de pointes (24).…”

Deacetylclivorine: A Gender-Selective Metabolite of Clivorine Formed in Female Sprague-Dawley Rat Liver Microsomes

“…Four selective P450 inhibitors [10 M pilocarpine to CYP2A1 (Kimonen et al, 1995;Lewis, 1998); 30 M diethyldithiocarbamate to CYP2E1 (Eagling et al, 1998); 5 M ketoconazole to CYP3A1/2 (Ghosal et al, 1996;Shayeganpour et al, 2006); and 100 M sulfaphenazole to CYP2C subfamily (Eagling et al, 1998)] and two carboxylesterase inhibitors (TOCP, a nonselective microsomal carboxylesterase inhibitor, and PMSF, a selective hydrolase A inhibitor) at various concentrations were used to study their effects on the microsomal metabolism of clivorine in female rats. The concentrations of individual P450 inhibitors utilized were chosen based on the published reports as indicated above, to achieve significant inhibitions of the corresponding isozymes in rat liver microsomes.…”

Carbonated soft drinks alter hepatic cytochrome P450 isoform expression in Wistar rats

“…Rat CYP3A2 and human CYP3A4 are involved in the metabolism of erythromycin, nifedipine, lidocaine, testosterone, aflatoxin B1 and benzo[a]pyrene (28,30). Furthermore, CYP3A4 is highly expressed in the adult liver and small intestine (30), and metabolizes xenobiotics and carcinogens (32,33), as well as numerous endogenous compounds, such as bile acids, cholesterol, prostaglandins, fatty acids, retinoids, leukotrienes and biogenic amines (32,34). The induction of CYP3A2 by SDC may be attributed to the presence of taurine, as it was demonstrated that taurine enhanced the induction of CYP3A4 by rifampicin in HepG2 cells (35).…”