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Dear all,
I'm working on an acetylated semisynthetic compound. This compound is stable in water and methanol. However, once dissolved in plasma for preparation of calibration curve for PK work, a substantial amount (20 -30%) of this compound breaks down to the parent compound. The break down can happen very fast, within a few minutes. If left longer in plasma, I believe this compound can totally convert to the parent compound. I tried PMSF, an esterase inhibitor to prevent the breakdown of this compound. There seems to be some improvements but inconsistency does exist. How could I solve this problem in order to build a reliable calibration curve? Any suggestion is welcome.
Thanking you in advance.
Michel Wong
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Dear Wong,
Ensure you are working at the optimum pH for the esterase inhibitor
activity. Check the PMSF stock solution also and remember it is not stable
in aqueous media. PMSF is fairly specific for esterases containing serine in
catalytic site. Maybe you could try a inhibitors mix.
Regards,
Prof. Dr. Diogo de O. Silva
Universidade Federal de Sao Paulo
UNIFESP - Campus Diadema
Rua Prof. Artur Riedel, 275 - Eldorado
CEP: 09972-270, Diadema, SP - Brasil
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Hi Michel,
Perhaps you could try to acidify the drug free plasma prior to spiking
it with your test drug.
You can try by the addittion of 10-20 uL of (50% citric acid in water)
per 1 mL of plasma. You should check first if your drug is stable at
that pH.
This is how I managed the instability problem of remifentanyl in plasma,
which is also a very good substrate of plasma esterases with a t1/2 of
only a couple of minutes.
Please report back if this proves to be successful.
Regards,
Jurij Trontelj, PhD
Faculty of pharmacy
University of Ljubljana
Slovenia
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Dear Michael,
Regarding breakdown of an acetylated compound in plasma, I am assuming from your message that it is an acetate ester. We had many of esters in development over the years, often as prodrugs, but acetates were very labile and were not advanced. If possible, consider other esters such as isobutyric acid, trimethyl acetic acid, valine, etc.
For esters in general, we never had adequate success in stabilizing them in blood or plasma by addition of esterase inhibitors (e.g., PMSF), either singly or in combinations. Additionally, such additives are typically quite toxic and require care in use and handling. We did have success (i.e., adequate stability to enable validation of methods according to FDA guidelines) by keeping all solutions very cold, thawing frozen solutions quickly to 4 C, and conducting the sample processing quickly. Once compounds were out of blood or plasma, they were almost always adequately stable in the extraction media (e.g., acidified acetonitrile or an organic solvent).
Hope this helps.
-Tom
Thomas L. Tarnowski, Ph.D.
Bioanalytical Development
Elan Pharmaceuticals, Inc.
800 Gateway Boulevard
South San Francisco, CA 94080
thomas.tarnowski.at.elan.com
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Prof. Dr. Diogo de O. Silva,
I prepared the PMSF stock in methanol. What are the inhibitors mix that you would suggest?
Michel Wong
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Dear Michel,
If its not practical to denature, deactivate and precipitate the plasma
proteins "upon sample collection", I would expect the validation to be very
difficult. Tom mentioned valuable points, but additionally, plasma from
humans versus mice (for example) can be expected to react somewhat
differently depending on your choice of inhibitors. Even if you succeed
with a cocktail of irreversible nanomolar esterase inhibitors in rodents,
you may not be able to use the same methods clinically.
Good luck with that!
-Shawn
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You can also try, Dichlorvos and Sodium Fluoride.
Thanks
Tausif
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You can try Sodium formate buffer at pH 4 along with sodium EDTA. pH plays a critical role in this assay and use formic acid to adjust the pH. NaF concentration between 6-8 mg/mL should be sufficient but may need to optimize the concentration of the NaF in plasma.
Two references related to ester hydrolysis are listed below.
1. The prodrug approach to better targeting, meeting report by Longqin Hu, Rutgers University School of Pharmacy, NJ, USA.
2. Preliminary in vitro and in vivo investigations on methylprednisolone and its acetate, Dyal C Garg et el, Research communications in Chemical Pathology and Pharmacology, Vol:22 No.1.
Hope this helps.
Regards,
Anila
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Dear Jurij Trontelj,
Thanks for your suggestion. I could have a try on this. However, i have very limited mice plasma.
Michel Wong
--
Dear Tom,
Thanks for providing me impressive ideas. Conducting the experiment on ice would be the easiest way among all the possible solutions.
Michel Wong
--
Dear all,
Thanks a lot for your valuable suggestions. I do appreciate them very much. Since i have very limited source of mice plasma, which method should i start out?
Michel Wong
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Dear Jurij Trontelj,
Can I know what is the concentration of stock solution for citric acid? I got citric acid in solid form. May I know what is the final pH by adding 10-20 uL of (50% citric acid in water) into 1 mL of plasma?
Thank you very much.
Regards,
Michel Wong
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