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Hello sir,
1. Can any one please tell us how to characterize the metabolites as
actives or inactives.
2. With out isolating and characterizing the metabolite , how to
quantitate without a standard.
3. The metabolites are generally polar when compared to actives, how
the extraction procedure of parent drug can be employed to the
metabolites.
We will appreciate the answers.
Thanking you
Ranjith
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The following message was posted to: PharmPK
Ranjith,
Aberra Fura at BMS has recently added an excellent review on
pharmacologically active metabolites to his list of other work on the
same subject:
Fura A, "Role of pharmacologically active metabolites in drug discovery
and development," **Drug Discov Today**, 2006 Feb;11(3-4):133-42.
In the paper, he describes in detail no less than five pieces of
experimental evidence for the indication of an active metabolite:
1) apparent disconnect between in vivo and in vitro pharmacological data
2) apparent disconnect between PD and PK data
3) higher pharmacolical response for extravascular dose vs. parental
dose
4) higher (or lower) ppharmacological response in presence of metabolism
modulators
5) unintended pharmacological response
As to your other question, by definition, quantitation requires the use
of a standard; however, if you make some assumptions (with caution):
equimolar absorption coefficients for the "parent" and metabolite if
using LC/UV and/or equimolar ionization if using LC/MS, then a
semi-quantitative approach can be made to evaluating drug levels.
As for your last question, you bring up a good point in extraction
efficiencies; indeed, we have seen times in our own lab when larger
ratios of organic in an extraction solvent that work well for the parent
compound do not efficiently extract polar metabolites.
Best Regards,
Jim Schmidt
Research Scientist, DMPK
Lexicon Genetics
The Woodlands, TX USA
jschmidt.-at-.lexgen.com
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The following message was posted to: PharmPK
Dear Ranjit,
>1. Can any one please tell us how to characterize the >metabolites
as actives or inactives.
Apart from indirect means mentioned by Jim Schmidt, direct way of
checking this is by conducting activity studies using metabolites
either synthesisied(if the structure is known and feasible)or
isolated from in vivo (urine/bile etc) or in vitro (microsomal
incubations etc) study samples using preparative HPLC.
>2. With out isolating and characterizing the metabolite , how >to
quantitate without a standard.
Quantitating metabolite without a standard is never going to be
accurate. But, there is a means to achieve fairly accurate data if
only single metabolite is formed and you have the in vitro system
that can generate it (eg. microsomal). What I siggest is to incubate
parent in the in vitro system such that parent is completely
metabolised and you see only metabolite (100% metabolite). Now by
analysing this along with control (100% parent) you can establish
parent to metabolite molar absorbance (UV) or ionisation (MS)
response ratio (response factor). Once you know this, you can use
parent to quantitate the metablite by using the above response
factor. However, you need to make sure you have a extraction method
that can extract both parent and metabolite with same efficiency -
which leads to your next question.
>3. The metabolites are generally polar when compared to >actives,
how the extraction procedure of parent drug can be >mployed to the
metabolites.
You can adopt double extraction method - first using a non polar
solvent mixture (suitable to extract parent) followed by a polar
solvent mixture (suitbale to extract metabolite). I know its tedious
but gives you good results.
Kasiram.
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The following message was posted to: PharmPK
Hi,
You can use 14C or 3H labeled parent for dosing, then
you can do HPLC/radio detection profiling to know the
metabolite percentage versus parent at each time
point.
Regarding the extraction recovery, if using the above
method for quantification, it is better to optimize
the your extraction condition to have reasonable
recovery for both the parent and metabolite. If you
have got the std of metabolite later. If the
metabolite is quite polar, maybe you need a separate
extraction methods for the parent and the metabolite,
respectively.
Hope this can help.
Xiaodong
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