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Dear all,
I am trying to calculated intrinsic clearance from in vitro metabolic
stability study in microsomes using substrate depletion approach.
While calculating rate of metabolism whether to take (Clast-C0)/(Tlast-
T0) or only initial rate of metabolism should be considered. Please
discuss rationale also.
Thanks and with best regards
Ravi
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The following message was posted to: PharmPK
Ravi,
To calculate intrinsic clearance from in vitro data it is recommended to
first establish conditions for linear kinetics (in terms of initial
substrate and protein concentrations). If your initial substrate
concentration is <= Km then you will not be saturating the enzyme and
you will usually have first order kinetics (of course, make sure NADPH
is saturating and therefore not rate-limiting). If you plot log [S] vs
time you can then fit a regression line to the linear portion and
calculate half-life based on this.
The initial rate is used as several factors may affect the rate of (in
vitro) metabolism over time such as product inhibition or general loss
of enzyme activity.
When comparing intrinsic clearance for several compounds (i.e. in a
small-molecule screening assay) it is important to standardize the
conditions. It all depends on the affinity of the individual CYPs
involved but with microsomes an initial [S] in the range 1 - 10
micromolar with a total protein concentration ~ 0.5 mg/ml is typical.
Depending on the circumstances, the effects of any non-specific protein
binding may also need to be considered.
Hope this is helpful - good luck!
Roy Turton
OSI Pharmaceuticals, Inc.
Boulder, CO 80301
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Dear Ravi,
While calculating rate of metabolism, initial linear conditions are
always preferred to reduce the underprediction of CLint.
In substrate depletion approach using microsomes, ideal conditions
would be enzyme concentration below 0.5mg/ml and incubation time less
than 30 min (at longer incubation time the microsomal enzymes may
loose their activity). This will reduce likelyhood of biphasic/
multiphasic depletion profile and underprediction of CLint by
decreasing the potential for microsomal binding and by maintaining
enzyme stabilty.
Hope it helps.
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