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The following message was posted to: PharmPK
Dear Members,
I have some peculiar data being generated in our microsomal assay. The assay is a: 60 minute time course (5pts), run in duplicate, a no-NADPH control group is also run in parallel (T0 and T60 only), 1mg/ml HLM (human liver microsomes),10uM compound (SIM limitations). The compounds are synthetic small molecules. I run a CTRL compound that works fine (T1/2 = 10-15min).
The issue involves turnover (up to 90%) of a few compounds for non NADPH added samples (so the non-P450 control group). Which in itself could probably be explained by other non-CYP enzyme (xanthine oxidase, aldehyde oxidase, mono-amine oxidase, etc.) but I am not seeing this degradation with the NADPH time course group.
My T0's for both the NADPH and Non-NADPH samples are very close (peak area ratio). The T60's are the ones that do not make sense. I have checked the samples table - so they have not been reversed (time course data anyway). PBS (with 5mM MgCl2, pH 7.4) stability data suggests that these compounds are stable (60 min, 37C).
Could this be a solubility issue ? Can you have data such that the P450 samples are stable whilst the non- P450 CTRLs are degrading?
A colleague suggested heat deactivating the HLM's to see if the decomposition is enzymatic but I'm still left with the question; why are these compounds stable with NADPH.
Any help/insights will be greatly appreciated.
-Ken
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The following message was posted to: PharmPK
Hi Ken,
In my experience, solubility issues are the most frequent cause of
"peculiar" data seen in this type of assay. However, this doesn't sound
like it is solubility related as your compounds are stable in PBS
(without protein) at a relatively high concentration. After ruling out
solubility I would check that there are no analytical issues.
A few questions:
Are the samples analyzed by LCMS? Do you add NADPH to the controls
post-stop reagent, for sample uniformity? What is the concentration of
NADPH in the samples?
I think it is worth running with heat-inactivated microsomes to confirm
that the degradation is enzymatic. It may be that a high NADPH
concentration is somehow inhibiting the non-CYP enzyme.
Regards,
Roy
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Hi Ken,
Few questions to resolve the issue-
a) do you terminate the non-NADPH reaction as well?
b) can you run NADPH control by omitting enzyme and terminate them as well?
Hope this will help.
regards,
Jignesh
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Did you scan un LC/MS for the new compounds formed? This could help to understand if it is a matter of non-CYP metabolism or just another trouble.
We had quite a different problem I would also like to share with you all.
It was not related to product stability but adsorption to polypropylene tubes and wells. As the affinity for HLM was higher than for plastic, there were no product loss in these samples but almost complete disappearance in PBS and non-HLM controls, that could be partially reversed by adding acetonitrile to the test tubes.
Regards,
k t
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The following message was posted to: PharmPK
Hi Jignesh,
1. Yes, all samples are terminated with 1:2 ratio of sample:organic (in this case, methanol/IS).
2. I see your point -NADPH seems to be the only difference in the two samples (by "omitting enzymes" - you mean heat deactivate the HLM's,I presume?). I can run this experiment in parallel.
How will I interpret the data?
Thank you very much for your response.
-Ken
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Hi Ken,
I mean, minus enzyme (no addition of enzyme) and with NADPH. And if you have heat deactivation procedure in place then it will be good.
This rule out possibilities of non-CYP mediated reactions and will be true control for actual reaction.
regards,
Jignesh
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