Back to the Top
The following message was posted to: PharmPK
Dear all,
We are trying to set up rapid plasma protein binding assay in our group.
I came across a device called Rapid Equilibrium Dialysis (RED, Thermo
Scientific). I appreciate if anyone from the group members would like to
share their experience on this or other similar product.
If you wish to discuss offline, please write to me at
(j.jaiswal.at.auckland.ac.nz).
Thanks,
Jagdish
Back to the Top
The following message was posted to: PharmPK
Hi Jagdish,
Good experience. The same issues as with regular equilibrium dialysis apply. Controls with PBS to assess binding to the device and stability of drug over time. The smaller size speeds up the achievement of equilibrium (deserves checking as well, despite assurances in the user guide).
Good luck, Jan
Back to the Top
The following message was posted to: PharmPK
Hi,
very good experince at 1 uM cmpd conc and equilibration times of 4 h. Parallel filling of the wells, analytics with a n LCQ-advantage. Very straight forward.
best regards
Dirk
Back to the Top
How about for sticky (severe NSB) compound?
Steven
Back to the Top
Hi Steve,
We had a very sticky compound recently. As long as you can document that you have reached equilibrium, it doesn't matter that e.g. 70% of your drug is device-bound. The ratio of your plasma-vs-PBS concentrations informs you about the protein binding. The only issue with device-bound drugs is that the concentrations in the compartments (especially the PBS one) become so small that your assay needs to be sensitive enough to quantitate these levels, while keeping the total plasma concentration within a clinically relevant range.
Jan
Back to the Top
Hi,
We use HTDialysis plates (http://htdialysis.com/) and it works fine for our compounds and commercial controls in the assay. Did anybody try the effect of shaking/proper agitation on the equilibrium time on the ht dialysis plates?
Thanks,
Raj
Rajinder Bhardwaj
DMPK
Lundbeck Research USA
Back to the Top
Hi all,
Regarding this issue, we have found some compounds having large non-specific binding (up to 95%) when disolved in buffer, but virtually no compound is lost when dissolved in plasma (i.e. more afinity to plasma proteins than to polypropylene). We don't really know how to deal with this, as we guess that in this case the concentration in the buffer chamber would be clearly understimated, and the % PPB would be overstimated.
Should we correct the buffer area/concentration taking into account the average % of non-specific binding? Would the equilibrium process correct this fact?
Many thanks in advance
Back to the Top
Hi all, as the systems seems to be back on business, I will try again my question:
Regarding this issue, we have found some compounds having large non-specific binding (up to 95%) when disolved in buffer, but virtually no compound is lost when dissolved in plasma (i.e. more afinity to plasma proteins than to polypropylene). We don't really know how to deal with this.
We guess that in this case the concentration in the buffer chamber would be underestimated, and thus the % PPB would be overestimated. Is that true or would the equilibrium process correct this fact?
Should we correct the buffer area/concentration taking into account the average % of non-specific binding?
Many thanks in advance
Back to the Top
The following message was posted to: PharmPK
As long as you reach equilibrium (do a time course to confirm), and your analyte by that time is not degraded, and your membrane not compromised by bacterial growth; your free concentration in buffer and your total concentration in the plasma side (you need to take that aliquot, don't assume it is the same as the starting concentration) are the concentrations you need. Another issue is what happens with your free concentration in the aliquot of buffer you take, while you suck it up into your pipet, which is plastic as well ;-) Nothing's easy.
Good luck, Jan
Back to the Top
The following message was posted to: PharmPK
Hi,
so you maybe want to check the vial surface, if you can handle all solution in plastic or glass (you can use inserts for HPLC-vials) you can prevent your compound attaching to pp-surface.
Dirk
Back to the Top
I think you can use the reverse dialysis method, you can make a aqueous solution of your compounds( make sure it can be detected by LC-MS), and make two dialysis bag with PBS and Plasma in it. the two bags were immersed into the aqueous solution of your compounds. after equlibrium, you can sample the outside and the inner bag concentration of drugs. then calculate the PB ration.
hope this help!
good luck
chang
Want to post a follow-up message on this topic?
If this link does not work with your browser send a follow-up message to PharmPK@boomer.org with "Rapid plasma protein binding assay" as the subject | Support PharmPK by using the |
Copyright 1995-2011 David W. A. Bourne (david@boomer.org)