Thummel and Wilkinson 1998). The majority of drug interactions of clinical
significance have occurred through interactions at the level of CYPs. Clinical
pharmacokinetic (PK) drug–drug interaction (DDI) studies have high cost and
are time consuming. As a consequence, there is a realistic limit to the number
and scope of clinical drug interaction studies that can be performed. The search
for alternative approaches to study drug interactions at early stage of drug
discovery has been encouraged. Thus, screening for the DDI usingin vitro
systems has become increasingly important for DDI predictions in man and for
more informed planning of clinical drug interaction studies.
For the inhibition assessment, a common strategy is to monitor effect of test
compound on the metabolism of CYP probe substrate using human liver
microsomes (HLM). Generally, most CYP-mediated reactions follow simple
Michaelis–Menten kinetics and their kinetic constants (KmandVmax) are easily
derived. If the addition of a CYP inhibitor results in inhibition of the enzyme
reaction, a value forKi(or IC 50 ) for reversible inhibition orKIandkinactfor
irreversible inhibition can be determined with suitable kinetic models. It is
accepted that the kinetic parameters can be used to predict and understand
in vivoPK and PD consequences caused by exposure to one or multiple drugs.
Among numerous CYP enzymes identified to date, six human hepatic CYP
isoforms (CYP1A2, 2C8, 2C9, 2C19, 2D6, and 3A4) play dominant roles in the
metabolism of clinical drugs, and these enzymes are commonly employed to
evaluate NCEs as inhibitors of the enzymes (Food and Drug Administration,
1997 and 1999). Many isoform-specific probe substrates have been recom-
mended by US regulatory agency (FDA) and Pharmaceutical Research and
Manufactures of America (PhRMA) (Bjornsson et al., 2003) and wildly used in
pharmaceutical industry.
In practice, enzyme inhibition is one of the easiest phenomena to measure in
a high throughput manner, as a result of experimental simplicity (monitoring
the effect of a test compound on the formation of metabolite(s) formed in
reaction of the CYP probe substrate with HLM, cDNA-expressed CYPs, or
hepatocytes). CYP inhibition evaluation is performed for one single isozyme or
multiple enzymes at a time. Various attempts have been made to increase the
throughput of these assays. Fluorescence-based assays for determining the
inhibition of CYP activity are available for a number of isozymes and are being
used in high throughput screening (HTS) of small molecules (Cohen et al.,
2003; Crespi and Stresser, 2000; Donato et al., 2004; Stresser et al., 2002;) In
these assays, the nonfluorescent substrate, upon enzymatic conversion,
generates a fluorescent metabolite that is detected in the fluorescence plate
reader. The assay is often performed with single endpoint measurement of the
reaction to make it easy to adapt to robotic systems. The most serious
drawback of this method is the interference from the intrinsic fluorescence or
possible fluorescence-quenching effects of compounds with the detection of
substrate metabolite. In addition, these compounds also lack the required
specificity as substrates for these enzymes, and hence cannot be used with
HLM but have to be used with individually expressed CYP enzymes.
514 ANALYSIS OFIN VITROCYTOCHROME P450 INHIBITION