Evaluation of Time-Dependent Inactivation of Cyp3a in Cryopreserved Human Hepatocytes

Abstract

ABSTRACT:Irreversible CYP3A inhibition by drugs constitutes one of the major causes of inhibition-based drug interactions. We evaluated timedependent inactivation of CYP3A in cryopreserved human hepatocytes for six structurally diverse compounds known to exhibit this property. Inactivation kinetic parameters were also determined using human liver microsomes. Except for diclofenac, which did not cause CYP3A inactivation either in microsomes or in hepatocytes at concentrations up to 100 M, time-dependent inactivation was observed in hepatocytes for amprenavir, diltiazem, erythromycin, raloxifene, and troleandomycin. The observed inactivation potency in hepatocytes (observed IC 50 ) was compared with the potency predicted using microsomal parameters (predicted IC 50 ). Despite satisfactory prediction for troleandomycin (1.35 and 2.14 M for the predicted and observed IC 50 , respectively), over-prediction of inactivation was observed for raloxifene, amprenavir, and erythromycin (observed IC 50 values 6.2-, 55-, and 7.8-fold higher, respectively, than the predicted IC 50 ). By contrast, the observed IC 50 for diltiazem in hepatocytes was approximately 4-fold lower than the IC 50 predicted from microsomal data (under-prediction). After correcting for factors including nonspecific binding and inactivator consumption, prediction was significantly improved for raloxifene (the observed IC 50 then became 2-fold higher than the predicted IC 50 ) and for amprenavir to a lesser extent. A specific P-glycoprotein inhibitor, 4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-N-[2-(3.4-dimethoxyphenyl)ethyl]-6,7-dimethoxyquinazolin-2-amine (CP-100356), modulated the observed CYP3A inactivation potency by erythromycin and troleandomycin. In summary, these studies reveal three important factors that must be considered when microsomal inactivation parameters are used to predict inhibition-based drug interactions in intact cell systems.The cytochrome P450 3A (CYP3A) family of enzymes is responsible for the metabolism of more than 50% of marketed drugs and some important endogenous substances. Consequently, drug interactions arising through the inhibition of CYP3A are of significant clinical importance (Lin and Lu, 1998). Mechanisms of CYP3A inhibition include reversible inhibition and time-dependent inactivation (TDI; also known as mechanism-based inhibition), and the latter has been recognized for many drugs and new chemical entities (Ito et al., 1998;Lin and Lu, 1998;Mayhew et al., 2000). Mayhew et al. (2000) showed that the inhibition of CYP3A by N-desmethyl-diltiazem, clarithromycin, and fluoxetine in humans was due to TDI instead of reversible inhibition. Traditionally, TDI is studied in human liver microsomes (HLMs) or cDNA-expressed enzyme systems to obtain inactivation kinetic parameters including the maximum inactivation rate constant (k inact ) and the apparent inactivation constant (K I ).These parameters, along with the expected inactivator exposure ([I]) and the degradation rate constant of the enzyme, which is usually ob...

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