Dear group,Back to the Top
We will attempt the first time to study in vitro metabolism of a particular
drug using human liver microsomes (from Gentest?). LC/MS will be used to
delineate the resulting metabolites. Would you suggest a pertaining procedure?
Thanks in advance,
Dear Viet,Back to the Top
Perform in vitro metabolism with controls (without Analyte) and have 0
minute and 30 or 60 minute samples. Run aqueous to conform the RT with a
gradient method (which you might be using on HPLC). Run control samples and
60 minute samples. substract the control from 60 minute sample. (if you
are having METID software, your can make it in no time). Get the
metabolite peak list and go for MS/MS on the same system (one again with
the help of METID software).
You can find lot many articles in DRUG METABOLIS AND DISPOSITION journal.
All the best.
Dr. Rajan, Ph.D.,
Ranbaxy Research Laboratories,
Dear Viet,Back to the Top
I wrote some time back:
"One of most common ways to investigate the contribution of phase I and
phase II enzyme involved in the metabolism of NCE is to carry out incubation
of NCE in microsomes/hepatocytes in the absence and presence of ABT (1 mM).
If you find significant depletion of NCE and inhibition by ABT that may
indicate involvement of P450s (phase I). If you observe, depletion of NCE
but no inhibition by ABT that would indicate involvement of non-P450 hepatic
enzyme(s). In this case, you have to identify the metabolite(s), if it an
oxidative metabolite, that would suggest involvement of enzyme such as FMO,
XO, AO, MAO etc. However, if it is a conjugated metabolite, you should
consider UGT, ST, NAT and other phase II enzymes."
Yes, you can use microsome. However, the advantage of using hepatocytes is
that these also have cytosolic proteins as well as you may find better phase
II enzyme activities (at least with fresh hepatocytes). Many pharmaceutical
companies are now routinely using hepatocytes for screening because of this.
Yes, you can get microsomes/hepatocytes from Gentest but there are many
other companies, including ours (www.CellzDirect.com).
I hope this help,
Kishore K. Khan, Ph. D.
8609 Cross Park Drive
Austin, TX 78754
Tel: (512) 615 2332
Fax: (512) 834 7767
Since you want to use LC/MS, keep the salt concentrations low. therefore,Back to the Top
all incubations should be carried in 50mM phosphate buffer or tris pH
7.4. Microsomal incubations are generally carried for 15-20 min. with 20
ug of protein in 200 ul total reaction volume and quenched with perchloric
acid 60%. We usually start the reaction by adding 1mM NaDPH. the range os
substrate concentration can be selected based on vmax plot. finally, once
the reaction is over you can either do the extraction and make up the
volume in your mobile phase or do the centrifugation at 14000 rpm for 10
min and take the supernantant for analysis.
Deepak,Back to the Top
You said that you start the reaction by adding 1mM NADPH. Is 1mM a final
concentration in reaction mixture or a concentration in stock solution?
If it is latter, what is the volume of NADPH you add? Thanks.
Thats my final concentration. I usually make the stock 10mM and use 20 ulBack to the Top
of that to start the reaction.
Dear Kishore:Back to the Top
Can you please provide a reference for your ABT suggestion so that I can
Dear Garcia:Back to the Top
Here is the reference:
Curr Drug Metab. 2003 Dec;4(6):527-34. Related Articles, Links
Reaction phenotyping in drug discovery: moving forward with confidence?
Williams JA, Hurst SI, Bauman J, Jones BC, Hyland R, Gibbs JP, Obach RS,
Department of Pharmacokinetics, Pfizer Global Research and Development, 2800
Plymouth Road, Ann Arbor, Michigan 48105, USA. James.Williams2.-at-.pfizer.com
For the pharmaceutical industry, one of the challenges in evaluating the
risk of future compound attrition at the discovery stage is the successful
prediction of the major routes of clearance in humans. For compounds cleared
by metabolism, such information will help to avoid the development of
compounds that will exhibit large interpatient differences in
pharmacokinetics via 1). routes of metabolism catalyzed by functionally
polymorphic enzymes and/or 2). clinically significant metabolic drug-drug
interactions, in the later stages of development. The degree of intersubject
variability that is acceptable for a drug candidate is uncertain in the
discovery stage where knowledge of other important factors is limited or
unavailable (i.e. therapeutic index, pharmacodynamic variability, etc).
Reaction phenotyping is the semi-quantitative in vitro estimation of the
relative contributions of specific drug-metabolizing enzymes to the
metabolism of a test compound. However, reaction phenotyping in the
discovery stage of drug development is complicated by the absence of
radiolabelled parent compound or metabolite bioanalytical standards relative
to later stages of development. In this commentary, some of the approaches,
based on published data, which can be taken to overcome these challenges are
discussed. In addition, knowledge of the molecular structure (i.e. specific
chemical substituents), physicochemical properties, and routes of clearance
in animals can all help in making a successful prediction for the routes of
clearance in humans. In combination, the objective of these studies should
be to reduce to a minimum the risk of finding significant inter-patient
differences in pharmacokinetics at a later stage in development due to
significant metabolism by polymorphic enzymes or drug-drug interactions.
Consequently, this data should be used to avoid costly late stage attrition.
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