Back to the Top
The following message was posted to: PharmPK
Hi Kavita,
You should express the concentration/gm of tissue for
this type of work. Do you know the relative BA of your
drug i.e by IV administration of drug in an
un-capsulated form. This would not apply if your
objective is to make an iv formulation of a poorly
soluble drug by nanotechnology. What type of tissue
solubilizer are you using? Most should solubilize the
polymer and tissue-that is why you are seeing a clear
solution. Check where you are losing drug- filtration,
binding to homogenization tubes etc etc. Do a spiked
tissue study with a cold or hot compound and see how
much you recover from each tissue you are interested.
Jagdish
Back to the Top
Thanks for your informed email, Jagdish!
I did a standard plot with each tissue such as [brain, liver, lung,
heart,spleen and kidney] by adding known concentration of radioactive
nanoparticles and getting a standard plot. Then I normalized the data
to concentration in microgram/mg of each tissue based on standard
plot that I obtained. However, I can recover only 10% collectively
from all organs analyzed.
You mentioned that I can calculate the recovery. But based on the
above info, how should I go about it? Might be sounding little dumb:(
I am using a tissue solubilizer [Scintigest] and analysing it witjh a
Toluene based scintillation cocktail.
I believe the polymer PLA is also getting solubilized coz many people
report using this protocol for nanoparticles.
But, the data analysis is what is baffling as I am not able to
account for 100% radioactivity bearing few losses in organs that I
havent harvested.
Your information has been very helpful!
Thank you so much!!!
Regards,
Kavita
Back to the Top
Dear Kavit,
Obtaining the % dose in a tissue/organ is the customary objective of
radio-ADME studies. I'll summarize this calculation as follows: Once
you have determined the specific activity of the radiolabeled
substrate in the tissue (DMP/g) by LSC and recorded the total weight
of the tissue/organ sample (g), you can multiply these together to
compute the total radioactivity in the tissue (DMP). You can then
multiply the initial specific activity of the labelled dose form by
the total tissue activity to obtain the total amount of radiolabelled
substrate in the tissue. Dividing this amount by the total dose x
100% yields the % of dose in the tissue.
The problem, as you rightly recognized, is that the radioactivity you
are measuring in the tissue digests comes from at least two and
possibly four sources: first, from the original encapsulated dose
form carried to the tissue, and second, from the labeled substrate
released either during or after deposition in the tissue. A third
source of radioactivity in the sample you are measuring could arise
from metabolites of the labelled substrate formed after its release
and prior to or after transport to the tissue. Another possible
contribution to the chemical nature of the radioactivity in your
digest could be tritiated water due to exchange of the tritium on the
substrate that might occur before or after metabolism.
You will need to do some experimenting with your sample in order to
determine the nature of the radioactive species present. This would
usually entail some radio-chromatographic techniques such as radio-
TLC, radio-HPLC. You would also need to work on isolating the
radioactivity in ways that discriminate between the free and
encapsulated substrate, for example by extraction or partitioning
tissue homogenates prior to any solubilization. If you do not
determine the nature of the radio-material you are measuring, then "%
of dose" as the quantification unit may itself be misleading. The
most lenient measure would be to call it "% of tritium activity
administered" - which implies nothing about the nature of the
chemistry of the tritium source, and more or less equates to a mass
balance or recovery calculation. But even this is often quite useful
and satisfactory for both revealing something about the tissue mass
balance and the kinetics of the original labelled substrate.
A simple way to determine the presence of tritiated water in the
animal is to do a small scale Dean-Stark distillation. This involves
adding some benzene to a few ml of urine and distilling the mixture
to recover a small volume of water. The benzene-urine (water) mixture
forms an azeotrope that distills at a lower temperature than water
alone. The water separates from the benzene in the distillate and can
be measured for any tritium content by LSC.
If you are going to be involved in this area for a long time, it
would be worthwhile to explore what can be gained by dual labelling
the dose form using tritium for the active ingredient and C-14 for
the nanocapsule. Even more elaborate, perform a whole body
radiography to view the whole body distribution of both isotopes in a
longitudinal microtome slice.
We have an Excel-based software program called "Metabase" that is
designed specifically for sample management and data calculations of
radio-analytical ADME/PK studies. The program can be explored further
at: http://www.summitpk.com/metabase/metabase.htm
Best wishes for success in your radiolabeled ADME research endeavors,
and I'd love to ready your publications as they become available, if
public.
David S. Farrier
/\ /\
SummitPK.com /\ / \ /\ / \
/ / / /\ / \
=================================================
David S. Farrier, Ph.D. Phone: 970-249-1389
Summit Research Services Fax: 970-249-1360
68911 Open Field Dr. Email: DFarrier.-a-.SummitPK.com
Montrose, CO 81401 Web: http://www.SummitPK.com
=================================================
Back to the Top
I agree that tritium exchange with water could be an issue,
particularly if you digest your tissues at elevated temperatures and
for long times and your label(s) is not stable under these
conditions. You could do control "digest" with just your labelled
material, i.e. without any tissue.
Another way of looking at tritiated water is to freeze-dry a sample.
Last but not least, have you done all the corrections correctly? For
example, quench correction etc. Again, running a control "digest"
without tissue may give some important information in this respect.
Are you using an appropriate scint. cocktail for your digest?
Kind regards,
Frederik Pruijn
Back to the Top
The following message was posted to: PharmPK
Hello Kavita,
For a mass balance study along with the tissues did you count the
radioactivity in the excreta? I am assuming you collected the urine and
fecal sample over the duration of the study and the tissues for tissue
distribution at the end of the study? The 10% recovery could only tell
you the extent of tissue distribution of radioactivity over the course
of your study and not the percent excreted in urine and feces.
Sandeep
PharmPK Discussion List Archive Index page
Copyright 1995-2010 David W. A. Bourne (david@boomer.org)