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Dear all,
I have a question regarding prediction of hepatic clearance using the
well-stirred model with intrinsic clearance derived from microsome
assays. I understand that this approach gives resonable predictions for
drugs metabolised via the cytochrome P450 system. However, is the
approach still valid if we observe (additional) NADPH-independent
metabolism in the in vitro assays (chemical instability can be
descarted). What are the caveats?
Thanks in advance, Andreas.
Dr. Andreas Lindauer
Modeling & Simulation and in vivo ADME
Dept. of Pharmacokinetics and Metabolism
R&D Center. Ferrer Internacional S.A.
Juan de Sada 32, 08028 Barcelona
alindauer-research.aaa.ferrergrupo.com
www.ferrergrupo.com
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the well stirred model probably may also take an account of the
disappearance of compound, when NADPh is supplemented and likely it
may also take into consideration the chemical instability of the
compound.. However, testing compound stability prior in various
matrices are encouraged.
Thanks and regards,
vijayabhargava.
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Dear Andreas,
The intrinisc clearance that you obtain from the in vitro incubation you
are using (microsomes, hepatocytes...etc) describes the overall
clearance of compound in your system and does not differentiate between
degradtation (chemical instability), nonspecific binding, CYP
metabolism, or other NADPH-dependent pathways. In order to account for
those non-metabolic routes of compound loss, you have to determine them
experimentally. One way to do that is to determine stability and
non-specific binding in inactivated microsomes or in microsomes in the
absence of NADPH.
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Dear Vijay, Parnali, and Yazen,
Thank you very much for your replies. Let me follow-up a bit on this
topic in order to clarify my point.
Parnali, unfortunately hepatocyte assays are not an option at an early
stage of discovery for a matter of resources.
Vijay, you wrote:
the well stirred model probably may also take an account of the
disappearance of compound, when NADPh is supplemented and likely it
may also take into consideration the chemical instability of the
compound..
Could you please point me to a reference where this was investigated?
Yazen, you wrote:
The intrinisc clearance that you obtain from the in vitro incubation you
are using (microsomes, hepatocytes...etc) describes the overall
clearance of compound in your system and does not differentiate between
degradtation (chemical instability), nonspecific binding, CYP
metabolism, or other NADPH-dependent pathways. In order to account for
those non-metabolic routes of compound loss, you have to determine them
experimentally. One way to do that is to determine stability and
non-specific binding in inactivated microsomes or in microsomes in the
absence of NADPH.
I understand that there are many ways to differentiate experimentally
between the different mechanism that cause drug concentration to decline
in vitro. Incubation without NADPH actually was what we did when we
observed drug loss for a couple of compounds. My question is more
related to the predictive value of this in vitro information. When there
is only CYP related metabolism one can calculate the in vivo intrinsic
clearance by scaling-up with the amount of microsomes per gram liver
(e.g. 32 mg/g_liver for humans) and correcting for non-specific binding.
I have doubts if it is valid to make the same extrapolation if
metabolism occurs via NADPH-independent pathways. IMHO in case of
chemical instability it will not be valid at all.
Best regards, Andreas.
Dr. Andreas Lindauer
Modeling & Simulation and in vivo ADME
Dept. of Pharmacokinetics and Metabolism
R&D Center. Ferrer Internacional S.A.
Juan de Sada 32, 08028 Barcelona
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Andreas,
You are perfectly correct and this is an issue which we will deal with
more often. As Medicinal Chemists understand the structural basis of
P450 metabolism, they are building models where oxidative metabolism is
no longer the driving force in clearance. Microsomal clearance
emphasizes P450 mechanisms and even in hepatocytes, it is often
difficult to capture the kinetics of Phase II metabolism. This is one
of the pushes behind some of the newer 3D models which take into account
hepatocytes and other cell types which might influence some of the
metabolic reactions through release of transcription factors etc.
Norman Huebert
J&J PRD LLC
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