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Can some one tell me the experimental design to determine the drug
plasma-protein binding interaction studies ?
I am using, Amicon, Centrifree Millipore columns. I am doing the
experiment like this :
10 mL drug ( 20 mg/mlconcentration ) + 190mL plasma incubate for 1 hr
at 37 \0x221EC and add to the Amicon column, spin at 1500 g for 30 minutes at
4\0x221EC in an ultracentrifuge and analyze the filtrate by HPLC . Similarly,
I did the blank study with just adding plain water to another column.
Compare the HPLC peak area (s) and calculate the % binding .
Is the volume of the drug and plasma are OK to use in the columns ?
Thanks !
Sreekanth
sgutala.aaa.hotmail.com
gs1738.-a-.yahoo.com
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Sreekanth,
I assume you meant micro-litres (uL) and not milli-litres (mL), as the
devices only hold <1.5mL? If this is the case, then while you can use
only 200uL of sample, it will be difficult as you can only filter
through the device 30% of the original volume. Any more than 30%
(~70uL) will disrupt the binding equilibrium and you will get incorrect
results. You really need to stop the centrifugation at various
intervals and check that you havent passed the 30% filtration point,
which will be hard to do with such a small volume. Just centrifuging
the device at 1500 g for 30 minutes is a guide (upper limits). There
are some documents on the amicon/millipore web site -they are hard to
find last I looked, so email me direct if you want a copy.
Why are you centrifuging at 4C? I would have though it best to
centrifuge at 37C (centrifuge chamber and rotor), just like you did for
the incubation, if your results are meant to be indicative of "real
life". You might also want to check that "spiked" plasma
concentration (that hasn't been filtered) using your HPLC assay too.
You should also confirm that there is no "non-specific binding" to the
device -this can be a real problem, especially with basic-amine
compounds, with these devices.
Regards,
David
David Foster, PhD
NHMRC Research Officer
Department of Clinical and Experimental Pharmacology
Faculty of Health Sciences
The University of Adelaide
Adelaide, South Australia 5005
Tel: +61 08 8303 5985
Fax: +61 08 8224 0685
Email: david.foster.aaa.adelaide.edu.au
http://www.adelaide.edu.au/Pharm/index.htm
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Sreekanth,
This technique does work, but you may wish to reduce your centrifuge
time.
The reason I say this is that you are significantly altering the sample
volume over a 30 minute spin. This in turn significantly alters the
protein
concentration in plasma. I would suggest centrifuging long enough to
analyze the filtrate, but not long enough to alter protein
concentrations in
the plasma.
Best of Luck
Mike
Michael D. Cameron Ph.D.
CellzDirect Inc.
8609 Cross Park Dr, Suite 100
Austin, TX 78754
Phone: 512-615-2286
Fax: 512-834-7767
michaelc.-at-.cellzdirect.com
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"Why are you centrifuging at 4C? I would have though it best to
centrifuge at 37C (centrifuge chamber and rotor), just like you did for
the incubation, if your results are meant to be indicative of "real
life". "
In our experience, centrifuging microcon devices at 37C causes an
unacceptable number of membrane failures. We use room temperature and
try to limit the centrifugation time to 10 min.
Another way to determine if you have a significant amount of non
specific binding (if you have a sensitive assay) is to collect a small
amount of the total ultrafiltrate (10%) in two serial aliquots and
compare the two results. Theoretically the concentration of the first
aliquot would be a lot lower than the second if binding was an issue.
Best regards,
Lori Payne, Ph.D.
Laboratory Manager
BASi Northwest Laboratory
3138 NE Rivergate Bldg. 301C
McMinnville, OR 97128 U.S.A.
phone: 503-472-8882 x207
fax: 503-472-4863
Yes David ! I meant it is micro litres (uL). I typed micro litre onlyBack to the Top
but for some reason, it has shown milli litre (ml). Millipore
technical people suggested me to spin at 4degrees at 1500g for 30
minutes or so. That is the reason, i have choosen 4C.
Can you please provide me the exact protocol that need to be followed
for plasma protein binding studies with amicon millipore columns ?
Thanks for the help
Sreekanth Gutala
sgutala.aaa.hotmail.com
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Sreekantha
The volumes of the drug and plasma are adequate; the plasma volume is
95% from the total volume which is acceptable, you do not want to
dilute the plasma more than 15%. However, and it depends on the
injection volume into the HPLC, 200ul total volume will give you ~60
ul filtrate's volume (30% from the total volume). If this is not
enough, increase the drug and plasma volumes keeping the same portion.
I could not understand your desired aim from the study; is it to
determine fb at specific nominal concentration or to determine the
binding parameters namely Km and Bmax?
In both cases you have to optimize your incubation time and assess the
following:
1. time needed for equilibrium between plasma protein(s) and your
compound
2. stability of the compound in plasma during the time of work
3. eliminate the non specific binding of the compound either to the
device or the membrane.
Last point is the temperature, you want to keep the temperature
constant at 37c during the experiment. Any change in the temp. will
change the binding affinity. If this is impossible, centrifuge the
sample(s) at room temp.
I hope that helps
Azmi Nasser
Azmi Nasser, B. Pharm., Ph.D. candidate
Department of Pharmaceutics
School of Pharmacy, Virginia Commonwealth University
410 N. 12th Street
Richmond, VA 23233
Phone: 804-828 8235
Fax: 804-828 8359
E-mail: anasser.-at-.hsc.vcu.edu
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