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Longtime reader, first time poster.
This list seems like such a helpful community, I'm hoping someone can help me out with a problem I
am having:
I was asked to characterize time-dependent inhibition of CYP1A2 by a new compound in human liver
microsomes. The problem is that the compound has a maximum aqueous solubility at pH = 7.4 of about 5
uM, a limit that is approximately the same as the expected affinity constant. Full KI/kinact values
are not necessary, but we do need to prove/disprove in vitro inhibition. Does anyone know of a
creative way to characterize TDI for low solubility compounds? Increasing inactivation time at low
concentrations would be ideal, but I am limited by enzyme system maximum incubation time (HLMs).
Thanks in advance!
-Justin
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Dear Justin,
This is a general problem with NCEs and solubility usually becomes the limiting factor for most
Invitro studies and unfortunately we have to live with it. Well I wonder if you have done two time
point data, if not please do that at 1 and 5 uM at two different time point may be 5 min and 30 min
in presence and absence of NADPH and see the ratio (presence of NADPH versus that in the absence of
NADPH). If this two point shows your compound do have TDI/MBI liability then it's worth to put
effort on calculating Ki/Kinact. I would also suggest once you are going for curve then go with step
down process (direct DMSO) not serial dilution and you can see the curve fall as indicative of
compound falling out may be the protein in buffer help in solubility and you may get higher
solubility of your compound compare to only PBS ph7.4.
Best of luck!
Zain
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If there are solubilty problem, you may dissolve the compound in
acetronitrile, methanol or DMSO. From my experience, avetronitrile is
interfere with CYP less than methanol. The final concentration of solvent
should be less 1 or 2%.
Wichittra
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Zain-
Thank you for your advice. If there isn't a way to get around the solubility issue, I suppose I
will check time and concentration dependency < 5 uM, as you suggested. Hopefully I'll be lucky...
Wichittra-
Thank you for your advice as well.I plan to dissolve it in DMSO first and dilute it into aqueous.
Hopefully the 1% DMSO will help the solubility compared to pure KPi buffer, but I anticipate that it
won't help enough...
-Justin
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