Evaluation of Luciferin-Isopropyl Acetal as a CYP3A4 Substrate for Human Hepatocytes: Effects of Organic Solvents, Cytochrome P450 (P450) Inhibitors, and P450 Inducers

Abstract

ABSTRACT:This study represents the first report on the characterization of luciferin-isopropyl acetal (LIPA) as a CYP3A4 substrate in human hepatocytes. LIPA metabolism by human hepatocytes was found to be linear with time up to 120 min and followed Michaelis-Menten kinetics, with apparent K m value of 15 M and V max of 41 pmol/ min/million hepatocytes for the hepatocytes used in the study. The nonspecific cytochrome P450 (P450) inhibitor 1-aminobenzotriazole (ABT) and the CYP3A4-selective inhibitor ketoconazole (KTZ) caused concentration-dependent inhibition of LIPA metabolism, with more than 50% inhibition observed at the lowest concentrations evaluated of 7.8 M (ABT) and 0.78 M (KTZ), and near 100% inhibition observed at higher concentrations. Substantially lower inhibitory effects were observed for the non-CYP3A4 inhibitors diethyldithiocarbamate, furafylline, omeprazole, orphenadrine, sulfaphenazole, and quinidine. The commonly used organic solvents-acetonitrile, dimethyl sulfoxide (DMSO), and methanolwere found to inhibit LIPA metabolism, with approximately 50% inhibition at concentrations of 5, 1.25, and 5% (by volume), respectively. The comparatively higher inhibitory effects of DMSO relative to that for acetonitrile and methanol on LIPA metabolism were consistent with its known CYP3A4 inhibitory effects reported by others. LIPA metabolism in human hepatocytes was found to be induced by the treatment of human hepatocytes with the prototypical CYP3A4 inducers rifampin, carbamazepine, omeprazole, phenobarbital, and phenytoin but not by the CYP1A2 inducer 3-methylcholanthrene. Although the selectivity toward CYP3A4 needs to be definitively evaluated using cDNA-expressed P450 isoforms, the results suggest that LIPA is a suitable substrate to be used with human hepatocytes for the evaluation of CYP3A4 activities.Cytochrome P450 (P450) is an important family of enzymes responsible for xenobiotic metabolism. Of the multiple isoforms, P450 isoform 3A4 (CYP3A4) is the most abundant of the isoforms in the human liver. CYP3A4 has been found to be responsible for the metabolism of a large variety of exogenous and endogenous substrates (Guengerich, 2006;Zhou, 2008). In drug development, there is a need to evaluate the inhibitory and inductive potential of drug candidates toward CYP3A4 to estimate their drug-drug interaction potential with the myriad drugs that are substrates of this important P450 isoform (Lin and Lu, 1998;Li, 2001).Quantification of CYP3A4 activity of an enzyme system (e.g., liver microsomes, hepatocytes) is performed by incubation with a CYP3A4-selective substrate, followed by quantification of the metabolites formed. The most commonly used substrates are testosterone and midazolam, with 6␤-hydroxytestosterone (Chauret et al., 1998;Easterbrook et al., 2001) and 1Ј-hydroxymidazolam (Emoto andIwasaki, 2007), respectively, as the CYP3A4-mediated metabolites (Guengerich, 2006). Quantification of these metabolites requires the use of high-performance liquid chromatography (HPLC), often facilitated by ma...

Assessment of Compound Hepatotoxicity Using Human Plateable Cryopreserved Hepatocytes in a 1536-Well-Plate Format

“…Further, Luciferin-IPA has been used to determine inhibition and induction of CYP3A4 in human hepatocytes. 42 Enzymatic activity of CYP3A4 was confirmed by calculating IC 50 values of the known specific inhibitor ketoconazole at 4 and 24 h after plating cryopreserved hepatocytes. The combined data from three of the donors provided average IC 50 values of 0.197 mM at 4 h and 0.146 mM at 24 h. Further, the CYP3A4 activity was sufficient to produce sigmoidal curves for the calculation of IC 50 values (Fig.…”

“…41,42 Cryopreserved hepatocyte lots IZT, LHO, and LMP were plated at 2K cells/ well as described above. At 4 and 24 h after plating, 14 ketoconazole concentrations with each point in quadruplicate (9.2 mM-1.1 nM) were transferred to the wells using the pin tool and incubated for 10 min.…”

A mechanistic insight into 3,4-methylenedioxymethamphetamine (“ecstasy”)-mediated hepatotoxicity

“…At present, there is a distinct lack of knowledge regarding the use of MDMA and the risks for DDIs, especially concerning interactions with PXR and/or CYP3A (Yueh et al 2005;Li 2009); despite the fact that there exists a significant clinical concern regarding the high prevalence of MDMA use and other recreational drugs consumption among some psychiatric patients (Modestin et al 1997). Many therapeutic drugs used not only for the treatment of psychiatric disorders but also clinical treatment of MDMA intoxications, such as diazepam (DZ), are (partly) metabolized by CYP3A (Spina et al 2003;Spina and de Leon 2007) or are agonists for PXR, such as the antiepileptic phenytoin (DPH) (Luo et al 2002).…”

“…The role of pregnane X receptor in CYP3A4-mediated MDMA bioactivation Since CYP3A4 appears to be a contributor to MDMA bioactivation, not only the interactions between MDMA and CYP3A4, but also with its main regulator pregnane X receptor (PXR) are of great relevance. CYP3A4 has a broad substrate specificity and is involved in multiple metabolic pathways such as regulation of steroid hormone homeostasis, bioactivation of environmental procarcinogens and anticancer drugs, including the metabolism of over 50% of the clinically used therapeutic drugs (Li et al 1995;Zhang et al 2003;Yueh et al 2005;Li 2009). Considering its relative hepatic abundance and its involvement in diverse pathways, CYP3A4 is considered as one of the major targets of adverse DDIs (Li et al 1995;Lehmann et al 1998).…”

Critical Human Hepatocyte-Based In Vitro Assays for the Evaluation of Adverse Drug Effects

“…We have previously introduced the use of human hepatocytes in P450 inhibition studies 20,34,35 . In the HTS human hepatocyte CYP3A4 inhibition assay described here, 384-well plates were used to reduce the quantity of hepatocytes, reagents, as well as the chemical to be evaluated 26 .…”