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Dear All,
We are working on CYP2C8 mediated metabolism of Rosiglitazone.
Incubations are done in human liver microsomes. We tried many protein
concentrations and incubation time, but unfortunately could not get
the Km value as its behaving linearly and not reaching saturation
phase. Although the literature says that Km should be close to 5 uM,
but unable to get it even at much higher conc. Could anybody suggest
what could be the reason or what modifications can be tried?
Best Regards,
Mansi
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The following message was posted to: PharmPK
Hi Mansi,
For Km determination we need to determine velocity of the reaction at
different SUBSTRATE concentrations NOT protein concentrations. Obtained
velocities against substrate concentrations should provide required
curve from which we can calculate Km and Vmax. Choose 3 to 4 half-log
substrate concentrations on either side of the expected Km for better
estimation.
Best regards,
Jayasagar Gundu
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Hi,
Are you sure that what you are looking at is the true metabolite peak?
This could make a big difference especially if it is a peak of a
degradation product of rosiglitazone.
Hope this helps,
Khalid Alkharfy
College of Pharmacy
King Saud University
Riyadh, Saudi Arabia
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Hi Mansi,
Can you provide details on the protein concentration, incubation time
and substrate concentration? I am assuming you are monitoring para
hydroxy rosiglitazone in the microsomal incubates. Is your analytical
method selective for ortho and para hydroxy rosiglitazone? May be they
are co-eluting together. This might still not provide an answer to the
fact that you are not seeing a Vmax at higher concentrations.
Ganesh Mugundu
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Hi,
One reason for not reaching the saturation phase for some of the drugs
is poor aqueous solubility of the drug at concentrations in excess of
the km value.
May be have a check with the solubility of the rosaglitazone.
Regards,
Raghav
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Hi Mansi,
for estimating the Km and V max, you should see for the linear
conditions. First of fix the protein then the time for the reaction,
this can be done by using reported Km. During this exercise check for
substrate depletion and metabolite formation ideally there should not
be large depletion (should be around 10-20%) and the metabolite be
quantifiable. Once you fix these parameters go for the Km curve by
taking 3-4 points on the lower side of reported Km and 3-4 points
after it. I hope i am clear and this is of some use to you.
--Naveed Shaik Abdul
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The following message was posted to: PharmPK
Dear All,
Thanks for your suggestions. But I am following the same concept where
you optimize time, protein first and then vary substrate conc for
determining km. Used 4-5 times half log sub conc on either side. But
would definitely check out whether the analytical method is able to
identify in para and ortho metabolite.
Thanks & Regards,
Mansi Sharma
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