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Dear group,
I have a compound whose solubility is around 2mg/ml.We have conducted
tissue distribution study in mice. The organs collected were liver,
lungs
and kidney which are highly perfused organs. Before collecting the
tissues
they were perfused with phosphate buffer. I have a doubt that by
perfusing
the tissues, the drug which has solubility around 2mg/ml, can drain from
the extracellular fluid along with buffer. So this might give
underestimation. Secondly, without perfusing the tissues, there could be
overestimation of tissue concentration. There are other methods like
microdialysis,Imaging techniques like PET and MRS and autoradiography
which
are not available in our lab.
Kindly, suggest any other possible method which would rule out
overestimation or underestimation of such type of compound.
Anasuya Patel
[If you measure blood concentrations and can get an estimate of the
amount of blood in the tissue you can correct for drug in blood in
each tissue. Many years ago we used radio-active albumin injected
just before killing the animal as a way of measuring blood in tissue
for this correction. There are probably other methods used by other
members of this listserv. Some simply blot the tissue which could
reduce the amount of blood in the tissue. - db]
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Once we did the tissue distribution study in rats.
We just blotted the tissue in the folded tissue/filter paper to avoid
maximum blood
component.
Hope this is the simple method to avoid the overestimation of the
analyte.
It worked.
Bye
Sripal
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Hi Anasuya, I am assuming that you are dosing the animal first invivo.
In that case, take a sample of blood from the heart just before
obtaining the tissues and then do a volume correction (alternatively,
you can take blood from the tissue blood vessels to be more accurate)
without flushing. Now I am again assuming that you can readily obtain
the vasculature volume for the organs in mice. I know there are
corrections values for cerebrovasculature in rats, but I am not aware
of such correction values for other tissues in mice , but I am
positive that you might get these values with some literature
searches (try searching physiologically based Pharmacokinetic model
papers).
Alternatively, in a control experiment if you can collect the
perfused buffer after perfusing at the blood flow of the liver for a
predetermined time (the blood in the organ being first all removed),
and analyzing to determine if any drug is present in the perfused
buffer. This is more time consuming and there might be some loss
which you might not be able to detect.
Indranil Bhattacharya
Principal Scientist
DMPK
GlaxoSmithKline
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Dear Anasuya:
You can also use published % blood volume in the
tissues/organs of your interest to correct for the
blood contribution. This method has its own
limitations and sometimes you may end up getting
negative numbers particularly at early time points.
For start you can look at:
Triplett, J.W., Hayden, T.L., Mc Whorter, L.K.,
Gautam, S.R.,Kim, E.E., and Bourne, D.W.A. 1985.
Determination of GalliumConcentration in "Blood Free"
Tissues Using a Radiolabeled BloodMarker. J. Pharm.
Sci., 74(9), 1007- 1009
Rostam
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Dear Anasuya Patel ? For tissue distribution studies, there appears
to be no easy biochemical or high throughput ?possible method which
would rule out overestimation or underestimation of such type of
compound.? Any useful approach probably needs to include
histochemical and microscopic techniques. Please let me know if
someone offers a reasonable and reliable single approach. There is a
long history about such efforts. I have been in it for several
decades. Here are some brief considerations regarding TISSUE
DISTRIBUTION STUDIES.
For meaningful distribution studies, AUTHENTICITY of in vivo
deposition as well as RESOLUTION of tissue and cells requires careful
attention:
1) The IN VIVO DISTRIBUTION of a drug and its metabolites needs to be
preserved in the procedure for the data to be an authentic
representation. And, we probably agree, an important component of
tissue distribution is related to specific receptor uptake and
retention. During tissue handling and preparation (e.g.,perfusion,
fixation), leaching and translocation artifacts are major problems.
Any step in the study that compromises authenticity should be
excluded. Studies of the intact organism and of unmolested tissues
would be ideal, but low resolution is the problem.
2)TISSUE HETEROGENEITY needs to be considered. It requires high
cellular resolution. Data from whole organs, chunks of organs, or
whole animals ? albeit providing robust statistics - may be
misleading. Target and non-target tissues frequently are
interdigitated, and sites of receptor binding with low capacity?high
specificity may remain hidden. If unspecific sites of high capacity-
low specificity deposition are mostly revealed, false negatives and
false positives regarding tissue distribution related to drug action
may be diagnosed. Also, non-invasive imaging procedures, while highly
attractive and yielding valuable information, frequently do not
provide enough resolution, - as is the case with the conventional
radioassays and whole-body autoradiography. In addition to these
common expedient low resolution methods - that may in fact be non-
expe dient, potentially misleading and therefore quite costly - ADME-
T investigations in drug development would benefit from the
occasional inclusion of an appropriate HIGH RESOLUTION approach.
We conducted comparative studies and evaluated different methods for
the tissue localization of drugs, demonstrating grave shortcomings
for radioassays and whole-body autoradiography if used alone,
contrasting with high resolution information for improved target
recognition, characterization, and target pharmacokinetics:
Stumpf WE. Drug Localization in Tissues and Cells. IDDC Press,
Chapel Hill, NC. 2003.
Stumpf WE. Drug localization and targeting with receptor microscopic
autoradiography. Journal of Pharmacological and Toxicological
Methods. 2005;51(1):25-40. Stumpf WE. The dose makes the medicine.
Drug Discovery Today. 2006;June, in press.
Walter E. Stumpf
Chapel Hill, NC
(www.walterstumpf.com)
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