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The following message was posted to: PharmPK
Dear all,
In a drug metabolism study using microsomes, I have to evaluate
kinetic parameters (Km and Vmax) from substrate consumption instead of
metabolite formation rates.
Could a standard approach be used (i.e. incubation of increasing
substrate concentrations, calculation of substrate depletion rate and
kinetics claculation from those data) or is the "in vitro T1/2 method
more reliable ?
Would appreciate any comments you have on the topic.
Thanks.
NP
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The following message was posted to: PharmPK
To determine Km and Vmax you can, in principle, run drug depletion.
Incubation at a low concentration (assumed to be << Km) will give you the
CLint - that's is the t1/2 approach. Incubation at a high concentration
will give you Vmax, this has obviously nothing to do with t1/2. As CLint
at << Km is equal to Vmax/Km, you can calculate the Km.
An additional second low concentration incubation (e.g. 2uM compared to
1uM) can be used to check if you are indeed under Km as the CLint values
should be the same in that case. See also a publication from us about
this: Xenobiotica 34, 229-241.
A more refined way is, as we use routinely, to use several drug depletion
concentrations and to fit the drug disappearance time-profiles to the
Michaelis Menten equation, using for example WinNonLin.
Hope it helps,
Ruben de Kanter PhD
Attrition Reducing Technologies
Nerviano Medical Sciences
Viale Pasteur 10
20014 Nerviano (MI)
Italy
Telephone +39 0331 58 1484
Telefax +39 0331 58 1105
ruben.dekanter.-a-.nervianoms.com
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The following message was posted to: PharmPK
Dear Nicolas
I think you can find what you want reading the
following paper:
Jones HM, Houston JB, Substrate depletion approach for
determining in vitro metabolic clearance: time
dependencies in hepatocyte and microsomal incubations.
Drug Metab Dispos. 2004 Sep;32(9):973-82
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