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Dear Group,
I have joined a pharma company as a trainee in DMPK
I am bit confused with the experimental conditions used in liver
microsomal stability
I have few questions
When a molecule is taken for stability what should be the the protein
concentration and the drug concentration used for performing the
studies, how to make a cut off in the concentrations of the drug and
protein and does it really depends on the therapeutic area we are
working how many time points are to be taken
I will be very thankful to one and all
Regards
Suma Singh
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Dear Suma Singh,
One can use 0.5mg/ml protein and 1uM of drug conc, minimum of 5 time
points.
I hope this is clear to you,
Regards,
Samiulla
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Dear Suma,
the following steps will be used for matabolic stability,
First determine the Km value and incubation time (both should be
linear with product formation)
Determine the protein concentration by keeping the above (i.e Km and
incubation time) constant and take the concentration which gives the
linear range i.e. the product formation is linear and no binding of
the drug to that protein level.
Usually the metabolic stability is done at 0, 30, 60 and 120 mins.
Yes it depends on the therapeutic area so if you know the Cmax of test
compound take drug concentration equivalent to that.
simar
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Dear Vijender,
You say, metabolic stability is done upto 120 minutes. Can you please
give reference for this.
thanks
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It depends on what you're measuring as metabolic stability. Are you
simply looking at the disappearance of the parent compound, then the
mentioned conditions of 0.5 or even 0.25 mg/mL with 1-5 uM drug
concentration will suffice. I would not extend the incubations beyond
30 minutes if possible with 1 mM NADPH as the cofactor. If you are
looking at the generation of metabolites then aup to 20 uM is
appropriate in certain cases depending upon the km of the substrate
and which CYP or other mixed function oxidase is involved. Taking time
points is always a good idea, 5 10, 20, and 30 minutes. There are some
ncie regerences by McGinnity et al from drug metabolisma nd
Disposition that will give you a ncie overview and some elaboorate
methods. There are autoxidative processes in the microsomal catalysis
that can result in non linear metabolism that one needs to consider
also..
Sanjeev Thohan
Director DMPK
Exelixis, Inc
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It depends on what you're measuring as metabolic stability. Are you
simply looking at the disappearance of the parent compound, then the
mentioned conditions of 0.5 or even 0.25 mg/mL with 1-5 uM drug
concentration will suffice. I would not extend the incubations beyond
30 minutes if possible with 1 mM NADPH as the cofactor. If you are
looking at the generation of metabolites then aup to 20 uM is
appropriate in certain cases depending upon the km of the substrate
and which CYP or other mixed function oxidase is involved. Taking time
points is always a good idea, 5 10, 20, and 30 minutes. There are some
ncie regerences by McGinnity et al from drug metabolisma nd
Disposition that will give you a ncie overview and some elaboorate
methods. There are autoxidative processes in the microsomal catalysis
that can result in non linear metabolism that one needs to consider
also..
Sanjeev Thohan
Director DMPK
Exelixis, Inc
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