Vaughn P. Miller

CYP3A4 allelic variants with amino acid substitutions in exons 7 and 12 : Evidence for an allelic variant with altered catalytic activity

To determine the existence of mutant and variant CYP3A4 alleles in three racial groups and to assess functions of the variant alleles by complementary deoxyribonucleic acid (cDNA) expression.

The use of heterologously expressed drug metabolizing enzymes--state of the art and prospects for the future.

The first report of the functional, heterologous expression of a mammalian cytochrome P450 (CYP) enzyme occurred more than a decade ago. In the intervening years, these expression systems have been optimized with regard to the specific requirements for production of catalytically active enzymes. In this review, we discuss the strengths and limitations of heterologously expressed enzymes as they affect in vitro drug metabolism studies. Emphasis is given to new applications (screens for CYP inhibition and novel enzyme mixtures) that have been enabled by high level, functional expression of CYP enzymes.

Fluorometric High-Throughput Screening for Inhibitors of Cytochrome P450

Abstract: Rapid screening for cytochrome P450 inhibitors is part of the current paradigm for avoiding development of drugs likely to give clinical pharmacokinetic drug‐drug interactions and associated toxicities. We have developed microtiter plate‐based, direct, fluorometric assays for the activities of the principal human drug‐metabolizing enzymes, CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, as well as for CYP2A6, which is an important enzyme in environmental toxicology. These assays are rapid and compatible with existing high‐throughput assay instrumentation. For CYP1A2, CYP2C8, CYP2C9, CYP2C19, and CYP2D6, the potency of enzyme inhibition (IC50) is consistent regardless of the probe substrate or assay method employed. In contrast, CYP3A4 inhibition for an individual inhibitor shows significant differences in potency (>300‐fold) depending on the probe substrate being used. We have investigated these differences through the use of several structurally distinct fluorescent substrates for CYP3A4 and several classical substrate probes (e.g., testosterone, nifedipine, and midazolam), with a panel of known, clinically significant, CYP3A4 inhibitors. The use of multiple probe substrates appears to be needed to characterize the inhibition potential of xenobiotics for CYP3A4.

Design and application of fluorometric assays for human cytochrome P450 inhibition.

Publisher Summary One important aspect of drug safety is the extent to which a new drug entity (NDE) causes metabolism-based pharmacokinetic interactions with co-administered medications. Therefore, potential drug-drug interactions may be tested for each enzyme of interest with a suitable probe substrate. The validity of these assays depends on the use of human enzymes and the probe substrate/assay conditions measuring the activity of the subject enzyme with a high degree of specificity. However, if enzyme mixtures, such as human fiver microsomes, are to be used probe substrate choices are more limited, as substrates with multiple pathways of metabolism are usually inappropriate. The chapter discusses some general considerations for conducting fluorometric cytochrome P450 enzyme (CYP) inhibition assays and then provides a general method for conducting the assay for several of the drug-metabolizing CYPs. Assays should be conducted under initial rate conditions formation of the metabolite should be linear with respect to enzyme concentration and incubation time, and the total consumption of the substrate should be less than 20%.