Back to the Top
The following message was posted to: PharmPK
Dear all,
I'm working in S9 stability.i selected two Blanks for the S9
stability study.
one blank contain cofactor (5mM glucose 6 phosphate + 5mM Mgcl2
+0.5mM NADP)
with deactivated S9 fractions.
The second blank contain no cofactor but active S9 fractions.
Both blanks and test control group contain drug.
my question is my drug is degrading in all the three incubation's ,i.e
2 blanks and test .
And both the blanks degrade the same compound differently.
please advise me how compensate the loss of drug in blank.
Back to the Top
The following message was posted to: PharmPK
Hi Vijay,
You may also want to try stability in buffer and cofactors...Just
incubate your drug for the same period of time of your incubation and
look for desapperiance of your drug. Also, you can try to stop your
reaction by addind twice as much of the solvent that you use for it.
That may contribute to bring your drug back into solution.
Hope it helps.
Damaris Diaz
Back to the Top
Hi Vijay
Are you measuring parent compound or metabolite? If you are
measuring parent compund, it is possible following things may happen:
binding of substrate to the surface of the vials and esterase-
mediated metabolism. If you know % difference between two blanks, you
can differentiate the above possibilities from one another.
hope this helps
satheesh
Back to the Top
The following message was posted to: PharmPK
Hi,
You may want to test compound stability in your buffer ( no protein
or cofactors ) and compares with compounds stability in Methanol.
Also, looking at the structure always help understand compound
stability and/or look at the compound product ion(s), if you are
running your analysis using the ion trap MS, this may give you some
hints about the changes might occurs to your compounds it could be
non enzymatic.
Regards,
Amr
Amr NourAlDeen, Ph.D.
Senior Scientific Group Leader
Drug Metabolism and Pharmacokinetics
Lexicon Genetics Incorporated
8800 Technology Forest Place
The Woodlands, Texas 77381-1160
Phone: (281) 863-3667
E-mail: anouraldeen.at.lexgen.com
Back to the Top
Vijay
Did you check the stability of the drug in an aliquot containing only
the buffer and the cofactors. it could be that the drug is unstable
in the buffer itself.
Manish Issar
Sandoz
Wilson, NC-27893
Back to the Top
The following message was posted to: PharmPK
Dear vijay
First and formost we should know that the drug is stable in buffer pH
7.4. (Third blank can be no cofactors and no S9 fraction)
There can be various reasons like the drug is not stable at 7.4 pH in
buffer or S9 fraction is not inactivated properly
There is also a possibility of non enzymatic metabolism
Hope this helps
Gurpreet
Back to the Top
The following message was posted to: PharmPK
Hello Vijay,
If it is the parent that you are monitoring, then what are you comparing
your parent area (in blanks and test sample) with. I suppose you must be
having a standard (in buffer or some organic solvent with no S9 and
cofactors). If that is the case, did you process your standard similar
to that of blanks and test sample.
Next thing is, did you notice any additional peaks or just reduction in
parent peak area. If you do see additional peaks, obviously your sample
is degrading. If you are not seeing any additional peaks, then it could
be the problem in extracting your drug from S9 matrix. When you have
very low recovery you tend to get large deviation in your extraction.
This could be the reason for different peak areas in your two blanks.
If you need any further input from the group, you need to provide
additional information such as:
1. Detection method that you are using - HPLC-UV or LC-MS/MS
2. What did you compare your blanks and test sample with (eg. a
standard)
3. How did you process your standard and other samples (blank & test)?
4. On what basis have you concluded that your drug is degrading- did you
actually see any additional peaks or by just monitoring parent peak
area.
5. Is your drug very lipophilic/have solubility problems in the
incubation buffer.
Good luck,
Kasiram.
Back to the Top
The following message was posted to: PharmPK
Dear all thanks for answering questions.
if the compound is not stable in the S9 buffer or pH 7.4,how should
i proceed further in my S9 stability studies, or how i should compensate
loss of drug in cofacter,buffer,and pH 7.4.
Vijay.
Back to the Top
The following message was posted to: PharmPK
Hi Vijay,
Are you using LC-MS? If you are using it, you may want to check where is
your drug going. In other words, if you do your stability in buffer and
you see lost of the drug, try to check if it's converting into something
else, by spontaneous degradation. If that's the case, you may also look
for other factors that are contributing to it, such as temperature,
protein content, drug concentration, % of solvent added into the
reaction, what solvent are you using to dissolve your drug... If not,
you can try adding the drug at last, starting your reaction when the
mixture is warm, it helps a lot to keep the drug soluble and available
to be metabolize.
Hope it helps,
Damaris Diaz
Back to the Top
Hi Vijay the first thing I would do is look at the structure of the
molecule (your drug) and see what could be the potential causes of
your findings (highly unstable). Then look up some references in the
literature (if available) of compounds behaving similarly or having
similar structure or chemical groups. This will help you narrow down
further hypothesis that you might want to try to test.
Also what is the difference in stability of the molecule in different
media. You have mentioned that your compound is unstable in buffer,
cofactor only media etc, but how much difference is there among these
groups. I would also double check on the silanized and esterase free
tubes as previously mentioned.
Indranil Bhattacharya
Ph.D candidate
Dept. of Pharmaceutical Sciences
State University of New York at Buffalo
Usa
PharmPK Discussion List Archive Index page
Copyright 1995-2010 David W. A. Bourne (david@boomer.org)