Sequential Metabolism Is Responsible for Diltiazem-Induced Time-Dependent Loss of CYP3A

Abstract

ABSTRACT:Kinetic parameters (k inact and K I ) obtained in microsomes are often used to predict time-dependent inactivation. We previously reported that microsomal inactivation kinetic parameters of diltiazem underpredicted CYP3A inactivation in hepatocytes. In this study, we evaluated the contributions of inactivation and reversible inhibition of CYP3A by diltiazem and its N-desmethyl (MA) and N,N-didesmethyl (MD) metabolites. In human liver microsomes, MA was a more potent time-dependent inactivator of CYP3A than its parent drug, with apparent k inact approximately 4-fold higher than that of diltiazem at a microsomal protein concentration of 0.2 mg/ml. MD did not inactivate CYP3A. Inactivation of CYP3A by diltiazem was dependent on microsomal protein concentration (25, 36, and 41% decrease in CYP3A activity at 0.2, 0.4, and 0.8 mg/ml microsomal protein, respectively, incubated with 10 M diltiazem over 20 min), whereas inactivation by MA did not seem to be protein concentration-dependent. MA and MD were reversible inhibitors of CYP3A with competitive K i values of 2.7 and 0.2 M, respectively. In cryopreserved hepatocytes incubated with diltiazem, time-dependent loss of CYP3A was accompanied by increased formation of MA and MD, with the MA level similar to its K I at higher diltiazem concentrations. In addition, the metabolites appeared to be accumulated inside the cells. In summary, timedependent CYP3A inactivation by MA seems to be the major contributor responsible for the loss of CYP3A in human liver microsomes and human hepatocytes incubated with diltiazem. These findings suggest that prediction of CYP3A loss based solely on microsomal inactivation parameters of parent drug may be inadequate.The calcium channel blocker diltiazem is extensively metabolized via multiple pathways including N-demethylation, O-demethylation, and deacetylation (Fig. 1). The demethylation pathways are mediated by cytochrome P450 enzymes primarily in the liver, whereas deacetylation is believed to be carried out by esterases in multiple tissues (Homsy et al., 1995;Sutton et al., 1997). N-Desmethyl diltiazem (MA) is the major metabolite that undergoes further N-demethylation process to form N,N-didesmethyl diltiazem (MD). It has been shown that diltiazem caused clinically significant drug-drug interactions by decreasing the elimination of substrates through inhibition of CYP3A (Jones and Morris, 2002;Jerling et al., 2005). The cause of inhibition has been attributed to both diltiazem and its metabolites. In human liver microsomes (HLMs), MA and MD appeared to competitively inhibit testosterone 6␤-hydroxylation with inhibition constants (K i ) of approximately 2 and 0.1 M (Sutton et al., 1997). However, reversible inhibition may not sufficiently explain the observed drug interactions given that plasma concentrations of diltiazem and its metabolites are lower than their respective K i values. Time-dependent inactivation (TDI) was also observed in HLMs for both diltiazem (Jones et al., 1999;Dai et al., 2003) and MA (Mathew et al., 2000)...

Sequential Metabolism of Secondary Alkyl Amines to Metabolic-Intermediate Complexes: Opposing Roles for the Secondary Hydroxylamine and Primary Amine Metabolites of Desipramine, (S)-Fluoxetine, and N-Desmethyldiltiazem

“…We conclude that formation of an MI complex with CYP3A4 in vitro and possibly in vivo is through the secondary hydroxylamine metabolite of diltiazem. In addition, we show that the primary amine metabolite protects CYP3A4 from MI complex formation by accumulating in solution and competing for active enzyme (K i ϭ 0.2 M) (Zhao et al, 2007). However, similar roles in vivo cannot be assigned to these metabolites because the fate of the primary amine and secondary hydroxylamine of diltiazem is unknown.…”

“…The concentrations of the secondary amines in the inactivation assays and estimates of reversible affinities for their respective enzymes were as follows: DES ϭ 50 M with CYP2C11 (IC 50 ϭ 5 M, data not shown), (S)-FLX ϭ 10 M with CYP2C19 (IC 50 ϭ 4.7 M in HLMs) (Di Marco et al, 2007), and MA ϭ 2 M with CYP3A4 (K i ϭ 2 M in HLMs) (Zhao et al, 2007). The secondary hydroxylamine substrate concentrations used were lower to facilitate data acquisition and curve-fitting: DESOH ϭ 1 M, (S)-FLXOH ϭ 5 M, and MAOH ϭ 0.5 M. The initial rates of the loss of enzyme activity caused by the secondary amines and secondary hydroxylamines were determined in triplicate in the presence and absence of fixed concentrations of the primary amines at their respective K i or IC 50 values.…”

Confidence Assessment of the Simcyp Time-Based Approach and a Static Mathematical Model in Predicting Clinical Drug-Drug Interactions for Mechanism-Based CYP3A Inhibitors

“…This finding may be attributed to several factors. For example, although metabolites of several inhibitors are potent CYP3A inhibitors (Mayhew et al, 2000;Wang et al, 2004b;Zhao et al, 2007), the parent compounds have to be metabolized first to inactivate the enzyme; therefore, it is difficult to distinguish whether the parent compounds or their metabolites are responsible for CYP3A inactivation. In addition, for most inhibitors used in this study, the effect of autoinhibition may not be significant at the doses studied and could sometimes be dealt with by applying a large coefficient of variation of dmd.aspetjournals.org the clearance parameter (e.g., ritonavir).…”

Effect of the CYP3A inhibitors, diltiazem and ketoconazole, on ticagrelor pharmacokinetics in healthy volunteers

“…Diltiazem is a calcium channel blocker used to treat angina pectoris and mild-to-moderate arterial hypertension19. This compound is an inhibitor of CYP3A activity20,21 and is classified as a moderate inhibitor of this enzyme10. The antifungal agent, ketoconazole22,23, also inhibits CYP3A24 and is considered to be a strong CYP3A inhibitor10.…”