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i have a question related to use of positive substrates in CYP
phenotyping studies of NCE.In our lab we=A0routinely conduct CYP
phenotyping studies to identify the CYPs involved in the NCE. About 9
human CYPS are used in the study. From regulatory point of view is it
require to use positive substrate for each enzyme to ensure the CYP
activity ? As we routinely do other CYP related studies (CYP inhibition
studies) and we know that enzymes have CYP activity.So my quary is when
we do CYP phenotyping study is it require to run positive substrates
every time. i will be happy to receive any response from regulatory
point of view.
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The following message was posted to: PharmPK
When you are performing your phenotyping checks are you working
with human hepatic microsomal fraction (or S9 or hepatocytes etc.) or are
you using recombinant cytochromes P450? If you are using
microsomal fraction with the same enzyme-selective inhibitors you
employ as positive controls in your inhibition assessment studies
then, as you acknowledge, you already have the information you need (I am
assuming that you run the positive control inhibition check with relevant
concentrations of approved inhibitors).
If you are using recombinant enzymes, and do not intend to
perform phenotyping with microsomal fractions, then you will have to assay
the activities of those recombinant enzymes so as to calculate the
relative activity factor. Otherwise, because of the known
discrepancies between the two systems, there is no way to perform a
quantitative assessment of the roles of each of the enzymes.
My own preference is to perform both "subtractive" (microsomal
fraction with enzyme selective inhibitors or antibodies) and
"additive" (recombinant enzymes) phenotyping. Correlation studies with a
panel of phenotypically and/or genotypically characterised
donors can also be useful on some occasions.
I hope that this helps.
All the very best,
Bernard Murray, Ph.D.
Research Investigator/Scientific Leader
Drug Metabolism, PCS, PPD, GPRD, Abbott Laboratories, Chicago, USA
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