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The following message was posted to: PharmPK
I'm seeking expert opinion on qualitative prediction
of in vivo metabolism based on in vitro metabolism
data, particularly 2D6.
I have a compound in discovery phase that shows
negligible turnover in human microsomes or
hepatocytes, based on parent disappearance. However,
when it was incubated in the recombinant CYPs,
including 3A4, 2D6, 2C8, 2C9, 2C19, 2E1, 1A2 and 1B1,
only 3A4 and 2D6 showed appreciable turnover based on
parent disappearance. After 15 minutes of incubation,
the % parent remaining is 45% for 2D6 and 88% for 3A4,
and almost 100% for all other isozymes. In addition,
further incubation showed that the two polymorphic
alleles, 2D6*10 vs 2D6*1, turned over the parent at
differential rates. The incubation condition for 2D6
was 1 uM compound and 15 pmol of recombinant 2D6 in
phosphate buffer with MgCl2 and NADPH.
During discussion with my colleague, I was adviced
that the recombinant 2D6 data was not necessarily a
good predictor for human in vivo metabolism, i.e. the
studies I described above does not mean that in vivo,
2D6 is gonna be the major metabolizing enzyme, because
weird things can happen in recombinant systems.
I'm not an expert in 2D6, but I hope someone in this
forum is. Could you please comment on if the
recombinant 2D6 study was appropriately conducted, and
that if such data can at least qualitatively predict
that 2D6 will be involved in human in vivo metabolism
to some extent, although the exact extent needs to be
further determined.
I'd appreciate your input.
Lilian
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The following message was posted to: PharmPK
Lilian,
Yes, it sounds as if your study has demonstrated that both CYP2D6 and
CYP3A4 have the potential to metabolise your compound. As you correctly
point out, the more difficult step is to determine the quantitative
contribution of each enzyme to metabolism in vivo.
Have you checked microsomal binding of the compound (binding to
recombinant systems is often lower)? Is your compound likely to be
permeable enough to enter the hepatocytes? Was the absolute recovery of
substrate from the microsomal assay good? Could your compound be an
enzyme inhibitor (direct or metabolism-dependent)?
If you have the resources it might be useful to look for metabolites
generated by the recombinant enzymes (either look in full scan or at
speculative masses for common oxidative changes). If you can see
anything then you may be able to monitor rates of formation (semi
quantitatively) during incubation with the hepatic microsomal fraction
and/or hepatocytes. This tends to be more sensitive than following loss
of parent. If you are successful you can then use enzyme selective
inhibitors such as quinidine (CYP2D6) or azamulin or ketoconazole
(CYP3A) to look at the relative contributions of the enzymes. Another
possibility is to try microsomal fraction from a single donor with high
CYP3A activity and see if this increases the turnover. If so, you may
be able to get an idea of the relative contribution of the two enzymes.
If you have to scale up from recombinant enzymes to
microsomes-equivalent then deriving relative activity factors for CYP3A
and CYP2D6 allows some compensation for the differences in abundance and
activity of the enzymes in the two systems.
From your original stability data, assuming that positive controls
worked, and assuming there were no unstable (chemically or in the LC-MS
source) metabolites then your compound sounds quite metabolically stable
so the contributions of CYP3A4 and CYP2D6 to clearance in vivo would
likely be low.
All the very best,
Bernard
Bernard Murray, Ph.D.
Senior Research Scientist, Drug Metabolism
Gilead Sciences, Foster City CA
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The following message was posted to: PharmPK
Dear Lilian,
I would like to share the following few comments.
1) If you have not done it yet, it might be helpful to have a look on
the following paper which details general consensus views:
Bjornsson et al, 2003, Drug Metab Dispos 31, 815-832
2) about the experiments you mention: the first isoform screening
should have been performed at 1 uM or less. Using a higher
concentration may lead to enzyme saturation and underestimation of its
relative role in the metabolism of the drug
3) You are correctly aware that recombinant CYP systems are optimized
systems. The experimental conditions you report seem fine. According
to me, the main interest of such experiment is to exclude significant
participation of all the isoforms that did not metabolize the drug in
recombinant systems. For the isoforms that did metabolize the compound
in such optimized conditions, the in-vivo significance of the reaction
remains to be established.
4) Some groups have developed approaches to extrapolate data obtained
with recombinant systems, e.g., with the use of Relative Activity
Factors: MG Soars et al, Br J Clin Pharmacol 2003, 55:175-181.
5) In the paper cited above in point 1 are presented the other
(classical) approaches to determine the weight of CYP2D6 in the
metabolism of your compound (e.g., use of CYP2D6 specific inhibitor in
microsomes or hepatocytes). The slow overall rate of metabolism will
be a limitation, however, as suggested by the data in hepatocytes
which you mentioned.
6) However, it also may be recalled that CYP phenotyping
investigations are worthy only if it is first demonstrated that CYP
metabolism plays a significant role in drug's clearance.
Best regards,
Frederic MASSIERE
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Li-
It seems your microsomes might be derived from a poor metabolizer
(even if pooled). Try another batch or if you are really inquisitive
you can genotype. Another thing you can try is use these source of
microsomes to test known compound metabolism and compare your test
compound to it. Recombinant enzymes mostly show higher turnover rates
compared to microsomes. But disappearance of 50% drug suggests its a
major pathway.
Deepak
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Re: Metabolized by 2D6 in vivo
Dear Lilian,
I agree with Bernard, using the rate of substrate disappearance is not
the good idea. Let's try to quantitate the metabolite formation
instead. If you use substrate disappearance rate , what you have to
consider is the concentration of substrate that you use. If you use
too high concentration , the rate of disappearance may not be
siginificance.
You may also need to check the metabolism of your compound using a
batch of microsomal preparations (about 10 microsomal preparations,
preparing from different liver samples).
Data from recombinant enzymes may not be useful to predict percentage
of contribution in vivo. Using microsomes and inhibitor approach is a
good approach.
Wichittra
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Dear Lilian
As others have already said the metabolite formation assays are more
trustworthy for characterising the kinetics of your compound; however
in the case of substrate depletion there are some conditions that
should be considered
http://dx.doi.org/10.1124/dmd.106.010777
As a matter of fact, it seems recombinant data are more useful than
HLM data to predict percentage of contribution in vivo if the related
InterSystem Extrapolation Factors (ISEF) values are considered:
http://www.informaworld.com/smpp/content?content=10.1080/00498250310001646353
Please note that the ISEF values depend on rhCYP system/supplier and
will change from one enzyme to another one. Two recent successful
applications can be found at:
http://dx.doi.org/10.1124/dmd.108.020446
http://dx.doi.org/10.1111/j.1365-2125.2007.03070.x
Regards
Masoud
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