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We would like to do a PK analysis of a drug following IV administration into
rats. This drug is known to get metabolized very fast when spiked into
rodent blood (in-vitro). We would like to withdraw blood from the carotid
artery at 1, 3, 5, 10, 15, 20, 30, and 60 minutes following IV
administration. We want to do it in two groups of rats. We seem to have
two options of bleeding pattern. In option No 1 we can withdraw blood from
Group # 1 at 1, 5, 15 and 30 minutes and from Group # 2 at 3, 10, 20, and 60
minutes (Cross-over pattern). In option No 2 we can withdraw blood from
Group # 1 at 1, 3, 5 and 10 minutes (initial time points) and from Group No
2 at 15, 20, 30 and 60 minutes (late time points). Can someone please
advise me which is the best option and whether this can be done in a better
way. Also, any safety precautions we must take to minimize sample
variations?
Thanks
Ananda
[Interesting question. I've used alternating times, option 1 in the
past. The belief is that each animal is sampled over the longer time
to be more representative of the 'total drug exposure' - db]
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[A few replies - db]
From: "Ramakrishna S."
Date: Thu, 4 Apr 2002 13:29:57 +0530
To: david.aaa.boomer.org
Subject: RE: PharmPK Bleeding times following IV dosing
Dear Dr Ananda
Option 1 seems to be logically the best. Any comments from others?
regards
Dr. Sistla Ramakrishna
Scientist
Pharmacology Division
Indian Institute of Chemical Technology
Hyderabad 500 007
INDIA
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From: "Madrid, Kathleen"
Date: Thu, 4 Apr 2002 08:40:06 -0500
To: david.aaa.boomer.org
Subject: RE: PharmPK Bleeding times following IV dosing
When I used to run rat PKs, we would do all the sampling time points from
each animal. This is acceptable if you take only ~200uL of blood and
replace the lost volume with saline. It's not difficult if you have
cannulated the rats and just allow the blood to drip from the tubing into a
microtainer tube. You definitely need two or three techs working on the
study to make sure all the time points get collected on time.
Good luck.
Kathleen Ministri Madrid
Vivarium Manager
MedImmune, Inc.
35 West Watkins Mill Road
Gaithersburg, MD 20878
(301) 527-4379
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From: Candice Kissinger
Date: Thu, 04 Apr 2002 12:05:43 -0500
To: david.-a-.boomer.org
Subject: Re: PharmPK Bleeding times following IV dosing
Ananda:
It's a little hard to answer this completely, because I don't know the
volume of blood you require for analysis, whether the analytical method
uses whole blood, plasma, or serum, and what size of rat you plan to use?
Let's assume it's a standard 250 g rat and that 75 uL of plasma will
suffice for your analytical method. For a first-time-in-animal study for a
compound like this, I would implant a catheter in the artery, hook up the
animal to the automated blood sampler, let him recover for 24 hr., load and
program the IV dose regimen for some time in the evening when nobody was
present, go home, and then automatically collect samples post-dose into
refrigerated vials at 1 min. and then every 5 minutes thereafter for the 60
minutes. This approach would give you the entire 60 min. profile from one
animal and remove the stress of handling/confinement from manual sampling
and associated changes in circulation. Blood at each time point is
replaced with sterile saline, so you can easily take the 1.95 mL of whole
blood required (150 mL whole blood per time point -> 75 uL plasma).
Candice Kissinger
In Vivo Sampling Laboratory
Bioanalytical Systems Inc.
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Regarding the suggestion from Kathleen Ministri Madrid in response to my
query on Bleeding following IV dosing. Is it necessary to replace the lost
blood volume (200 ul per sampling) with IV saline, specially when we will be
bleeding at very short time intervals of a few minutes inbetween sampling.
Does not the drug concentration in the blood get diluted with saline and
provide a low plasma concentration in the subsequent sampling?
Thanks
Ananda
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With regards to some of the replies about replacing blood sample
volumes with saline, I would just like anyone's comments about the
possible advantages, disadvantages, etc. about two alternatives:
1. If assaying plasma: to spin down the samples immediately after
collection, pull off the plasma, and resuspend the pellet in a volume
equal to the aliquoted plasma, and administer to the animal (of
course the collected samples would have to be collected with
anti-coagulant)
or
2. Simply use blood from a naive animal to replace the sampled volume.
The second alternative seems more appropriate in this instance where
the sampling time points are very close to each other. Plus, since
their in vitro studies show extensive metabolism in spiked blood,
wouldn't maintaining the correct composition of blood be advantageous
over diluting it with saline? I guess the bottom-line general
question is: If PK is strongly influenced by plasma protein binding,
RBC uptake, etc. could there be a significant effect on overall PK if
saline vs. blood is used as a replacement when many samples are taken
from such small animals???
[Of course, the replacement of sampled blood volumes is much more of
an issue when taking a large number of samples from the same, small
rat. If large animals (>300g) are used and the total volume
collected is <2ml, replacing blood volume may not even be necessary
(and even some of the volume is inadvertently replaced when flushing
the cannula). ]
Regards,
Dave Jaworowicz
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You had better be careful about the rate of bleeding. You
may put your rats into
shock if you bleed them at too rapid a rate. Then your apparent
pharmacokinetic
disposition will in fact be compromised by hypovolemic shock. A
small rapid bleed is an
old tactic to induce short-term shock for physiological hemodynamic
studies. You also
need to limit you total blood volume sampled to less than 10% of
total circulating volume.
Saline will not remain in the circulation. It is essentially a
futile gesture to attempt to
replace blood volume with saline.
Dan Sitar, University of Manitoba
Daniel S. Sitar, PhD
Professor and Head
Department of Pharmacology and Therapeutics
sitar.at.ms.UManitoba.ca
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Dear Ananda:
The interesting question is - why do you want to use 2
different strategies in 2 groups of rats? Perhaps it is because you
want all the sampling times, but that is too much blood for a single
rat? Also, why do you want to use the specific times you have stated?
What you might consider is to use option 1, which I would
prefer, as then you are covering basically the same time period in
both groups, but not to commit your entire group of rats to either of
these protocols.
You might do something like 3 or 5 rats in each group, and
then make a population PK model of your findings. Based on this, and
the parameter values you find, use something like D - optimal design
to reposition your optimally informative time points for the results
you have found. You will have one optimal time for each parameter
value in your model.
What kind of model do you plan to make? How many parameters?
For the model you have in mind, D - optimal design, or one of its
variants, can give you information about the times at which changes
in the PK parameter values you have found will cause the greatest
changes in the serum concentrations. These are the times when you can
best "see through" the uncertainties in the environment that surround
the rats and best perceive their parameter values. This way you don't
waste your rats by locking yourself into some fixed protocol before
learning what is going on. You can repeat this procedure every 3-5
rats or so. Usually the population parameter values, and thus the
sampling strategy, will settle down after about 20-25 subjects, and
you will have optimized your sampling strategy by learning about the
population as you study it. For software, you can use the very good
optimal sampling module in Dave D'Argenio's ADAPT II software.
Best regards,
Roger Jelliffe
Roger W. Jelliffe, M.D. Professor of Medicine, USC
USC Laboratory of Applied Pharmacokinetics
2250 Alcazar St, Los Angeles CA 90033, USA
email= jelliffe.-a-.hsc.usc.edu
Our web site= http://www.lapk.org
PharmPK Discussion List Archive Index page
Copyright 1995-2010 David W. A. Bourne (david@boomer.org)