Effect of Albumin on Human Liver Microsomal and Recombinant CYP1A2 Activities: Impact on In Vitro-In Vivo Extrapolation of Drug Clearance

Abstract

ABSTRACT:Long-chain unsaturated fatty acids inhibit several cytochrome P450 and UDP-glucuronosyltransferase (UGT) enzymes involved in drug metabolism, including CYP2C8, CYP2C9, UGT1A9, UGT2B4, and UGT2B7. Bovine serum albumin (BSA) enhances these cytochrome P450 and UGT activities by sequestering fatty acids that are released from membranes, especially with human liver microsomes (HLM) as the enzyme source. Here, we report the effects of BSA on CYP1A2-catalyzed phenacetin (

A comprehensive assay for nine major cytochrome P450 enzymes activities with 16 probe reactions on human liver microsomes by a single LC/MS/MS run to support reliablein vitroinhibitory drug–drug interaction evaluation

“…However, more studies from this and other laboratories, recently, have been demonstrated a significant decrease in the Michaelis-Menten constant (K m ) of several CYP enzymes substrates (e.g. phenacetin of CYP1A2, paclitaxel of CYP2C8, tolbutamide of CYP2C9) depending on the addition of BSA to incubations on HLMs by sequestration of inhibitory long-chain unsaturated fatty acids released from membranes during the course of an incubation, especially with HLMs as the enzyme source (Ludden et al, 1997;Rowland et al, 2008;Tang et al, 2002;Wattanachai et al, 2011Wattanachai et al, , 2012. But, because of the nonspecific protein binding to albumin, a 2-fold effect of BSA was then suggested to accurate the kinetic data of CYP substrates: first, facilitation of the reaction via a decrease in the K m ; second, a decrease in Figure 6.…”

“…Given that the albumin effect was proposed to arise by sequestration of inhibitory long-chain unsaturated fatty acids released from membranes during the course of an incubation, especially with HLMs as the enzyme source according to previous references (Manevski et al, 2011;Rowland et al, 2007Rowland et al, , 2008Tang et al, 2002;Wattanachai et al, 2011Wattanachai et al, , 2012, we investigated the effects of BSA (0.5-2%, w/v) on the 16 probe reactions in pooled HLMs for the first time. As summarized in Figure 4, a significant increase was observed in the majority of reactions, while a reverse occurred in the hydroxylation of diclofenac, midazolam, testosterone and the oxidation of nifedipine when BSA was present.…”

Identification and Characterization of Psoralen and Isopsoralen as Potent CYP1A2 Reversible and Time-Dependent Inhibitors in Human and Rat Preclinical Studies

“…Hence, in the present study, the inhibitory effects of PRN and IPRN on CYP1A2-mediated metabolism were assessed in vitro and in vivo in rats and in vitro in humans using phenacetin as the probe substrate. A recent study reported that including bovine serum albumin (BSA) in CYP1A2 microsomal incubation resulted in enhanced CYP1A2 activity by reducing the apparent K m values that mainly attribute to the quenching of inhibitory effects by long-chain fatty acid residue in the HLM preparation by BSA (Wattanachai et al, 2012). Since including BSA in microsomal incubation has not become a standard practice in the pharmaceutical industry, the addition of BSA was not applied in this study.…”

Optimized Cocktail Phenotyping Study Protocol Using Physiological Based Pharmacokinetic Modeling and In silico Assessment of Metabolic Drug–Drug Interactions Involving Modafinil

“…The interaction (‘inhibitor’) profile was created based on published in vitro microsomal inhibition and hepatocyte induction data for CYP 1A2, 2C9, 2C19, 2D6 and 3A4 (Robertson et al, 2000). The presence of endogenous fatty acids in microsomal preparations is known to effect the in vitro determination of kinetic parameters for multiple CYP enzymes including CYP 1A2, 2C9 and 2C19 (Rowland et al, 2008; Wattanachai et al, 2012, 2015). As such, published K i values for CYP 1A2, 2C9, and 2C19 were scaled based on the known effects of endogenous fatty acids on these enzymes.…”