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The following message was posted to: PharmPK
Hi
I am planing on doing a PK study (plasma conc vs time profile) using
125 iodine tagged peptide. The end goal is to obtain area under the
curve in brain and blood after iv and intranasal administration in
order to fin the drug targeting index. However, I am concerned that my
peptide might get cleaved in the blood and I might detect the fragment
attached to the radioactive probe as a false positive. Any suggestions
will be greatly appreciated.
Thanks
Ashwini
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The following message was posted to: PharmPK
Dear Ashwini
One of the options may be studying the in-vitro stability of
radioactive tag in blood prior to study in vivo
Nadeem Irfan Bukhari
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Dear Ashiwini,
As Nadeem mentioned, you could study the plasma stability of the
radiolalled peptide first to check if your peptide is intact or not.
1. Incubate your radiolabelled peptide in mouse blood (or human blood
if available) for ... hours (decide how long is appropriate). Analyse
on electrophores gel (SDS PAGE) and evaluate radioactivity
distribution on for example a PhosphorImager (if intact peptide then
you have one peak, if fragments then you have several peaks). Don't
forget to include pure iodine-125 as reference
2. Another way is to inject your radiolabelled peptide in one mouse,
collect blood after .... hours and analyse as above.
Best regards,
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The following message was posted to: PharmPK
Ashwini,
If you have a meaningful measure of activity of your peptide, e.g.
cell-based signaling assay, you may consider using that to reassure
yourself that you are in fact tracking 'active' peptide. Of course,
this only assures you that a portion of the molecule is intact. A
sandwich ELISA, for example, may be useful if your molecule is large
enough, as the capture and detection areas are typically quite far apart
in the protein sequence.
Additionally, check your iodinated peptide in the assay to ensure an
"active" drug prior to studying it in vivo.
Warm Regards,
Mike Dodds
--
Mike Dodds, PhD
PK/PD, Pre-clinical Development
ZymoGenetics, Inc.
1201 Eastlake Avenue
Seattle, WA, 98102, USA
Email: doddsm.aaa.zgi.com
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The following message was posted to: PharmPK
Dear Nadeem, dear Ashwini
while this is certainly a helpful prerequite, it will tell Ashwini only
a part of the truth. According to Geissler F et al. Cancer Research
1992;52:2907-2915 and Stein R et al, Cancer Res 1995;55:3132-3139,
deiodination to iodide does hardly take place on the level of the
protein or peptide (provided one of the methods for the more stable
iodination has been used), but on the level of iodinated tyrosine. In
other words: the peptide/protein seems first to be cutted to amino
acids, and the iodinated tyrosine (mono- or di-iodinated) are then
deiodinated or potentially itself excreted in urine. Iodination of
histidine seems to resist these deiodinases.
Best regards,
Dietrich
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