Comparative Analysis of CYP3A Heteroactivation by Steroid Hormones and Flavonoids in Different in Vitro Systems and Potential in Vivo Implications

Abstract

ABSTRACT:A systematic analysis of the heteroactivation of CYP3A-mediated carbamazepine 10,11-epoxidation has been investigated in three different in vitro systems, namely recombinant CYP3A4 and CYP3A5, human liver microsomes (HLMs) and cryopreserved human hepatocytes. The effect of 10 endogenous steroids and flavonoids was studied over a range of substrate and effector concentrations. A novel heteroactivation model was used to obtain the parameters EC 200 (concentration of effector required to produce 200% control) and heteroactivation ratio (the ratio of maximum observed reaction velocity to control). The EC 200 values obtained in HLMs and human hepatocytes were corrected for nonspecific binding. Heteroactivation of CYP3A5 has been demonstrated with mean heteroactivation ratios in CYP3A5, HLMs and hepatocytes on average 2-fold greater than in recombinant CYP3A4 for most of the effectors investigated. In recombinant CYP3A4, heteroactivation was greatest at substrate concentrations below K m . Heteroactivation increased with effector concentration in a nonlinear manner and differed between effectors (mean heteroactivation ratios varied up to 12-fold). A greater extent of heteroactivation was observed in HLMs than in human hepatocytes for steroid effectors, but the opposite was true for flavonoid effectors. The observed heteroactivation of CYP3A in intact cells supports an in vivo relevance. From the in vitro heteroactivation data, a significant increase in clearance in vivo was predicted for substrates with a high dependence on CYP3A4 to the overall elimination, indicating that heteroactivation of CYP3A may be a potential source of interindividual variability.Cytochromes P450 (P450s) 3A are the most abundant P450 enzymes in the human liver and small intestine (Paine et al., 2006). In vitro assays of CYP3A-mediated metabolism are used routinely in the drug discovery effort to quantitatively predict in vivo pharmacokinetic parameters. However, these estimates may be confounded by atypical (non-Michaelis-Menten) kinetics such as autoactivation (homotropic cooperativity, evident as a sigmoidal kinetic profile), heteroactivation (heterotropic cooperativity, the activation of a substrate's metabolism by a separate effector compound), or substrate inhibition (inhibition of a substrate's own metabolism) (Houston and Galetin, 2005). A number of studies have addressed the issue of atypical kinetics (Tang and Stearns, 2001;Davydov et al., 2007). Heteroactivation is displayed by a number of CYP3A substrates in vitro, including the antiepileptic, carbamazepine (Nakamura et al., 2002;Egnell et al., 2003a). Heteroactivator-enzyme interaction has been rationalized either by the simultaneous binding of substrate and effector molecules (Shou et al., 1994) or assuming the existence of a separate effector-binding site (Houston and Galetin, 2005).Evidence exists that heteroactivation of CYP3A may occur in vivo. Coadministration of quinidine resulted in an acute increase in diclofenac clearance in monkeys, an effect also observed in m...

Substrate-dependent modulation of the catalytic activity of CYP3A by erlotinib

“…Moreover, CYP3A4 and CYP3A5 are the most abundant members of the CYP3A subfamily. A recent study has shown significant differences between the heteroactivation potential of CYP3A4 and CYP3A5 for CYP3A-mediated carbamazepine 10,11-epoxidation [43] . Inhibitors of CYP3A usually have different potencies for inhibition of CYP3A4 and CYP3A5 in terms of both reversible and irreversible inhibition [44,45] .…”

Heterotropic Activation of the Midazolam Hydroxylase Activity of CYP3A by a Positive Allosteric Modulator of mGlu5: In Vitro to In Vivo Translation and Potential Impact on Clinically Relevant Drug-Drug Interactions

“…However, the intrinsic enzymatic activity of the cytochrome P450 (P450) family, including CYP3A4, has also been reported to be significantly affected in vitro (and, in only a few examples, in vivo) through homotropic or heterotropic activation of the enzyme (Hutzler and Tracy, 2002;Obach, 2012). A wide variety of compounds and known drugs have been shown to be activators of CYP3A4 enzymatic activity, including 7,8-benzoflavone (Shou et al, 1994), quinidine (Ngui et al, 2000), and steroids (Henshall et al, 2008). The activation of CYP3A4 metabolic activity in vivo may represent an acute DDI scenario that could increase the clearance of a parent drug and/or increase levels of circulating, and perhaps toxic or active, metabolites.…”

Drug Interaction of Efavirenz and Midazolam: Efavirenz Activates the CYP3A-Mediated Midazolam 1′-Hydroxylation In Vitro

“…Activation (heterotropic positive cooperativity) occurs when P450 activity for a substrate is increased in the presence of another drug (Hutzler and Tracy, 2002) and may result in a change from hyperbolic Michaelis-Menten kinetics to nonhyperbolic kinetics (Atkins, 2005). Examples of CYP3A4 activators include 7,8-benzoflavone (Shou et al, 1994), quinidine (Ngui et al, 2001), and steroids (Henshall et al, 2008).…”