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Could someone please direct me to any relevant literature for formal
'serum stability' protocols to evaluate/ screen a series of esterified
pro-drugs, please?
Are there any
FDA approved regulatory guidelines for such experiments, like those
available for in vitro metabolism studies?
Many Thanks
Simon
Simon Clark, M.Sc.
Analytical Pharmacology
Antisoma Research Laboratories
St Georges Hospital Medical School
Cranmer Terrace
London
SW17 0QS
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The following message was posted to: PharmPK
Hi Simon,
This was posted on PharmPK a while ago:
On 13 Oct 2003 at 15:25:03, vijay upreti
(vijayupreti.at.yahoo.com) sent the message
Hello Fererica,A simple protocol to be followed for
determining in vitro stability inplasma, is to spike freshly
collected plasma with three concentrations,usually LowQuality
Control (LQC),Medium (MQC) and High (HQC). Thespiked plasma
samples are incubated at 37 Deg Centigradein a shakerbath
(100 rpm) and plasma samples collectedat serial time points
uptoa period of 6h and immediatelyprocessed and analyzed.Its
advisable tosimilarly spike and anlayze either water or
methanol as control.Good luck
vijayVijay V. Upreti.Department of Pharmaceutical Sciences
School of Pharmacy
University of Maryland. Baltimore. MD 21201.
You might want to look at these couple of articles on
bioconjugates too for simpler protocols:
1: Luo Y, Ziebell MR, Prestwich GD.
A hyaluronic acid-taxol antitumor bioconjugate targeted to
cancer cells.
Biomacromolecules. 2000 Summer;1(2):208-18.
PMID: 11710102 [PubMed - indexed for MEDLINE]
2: Luo Y, Prestwich GD.
Synthesis and selective cytotoxicity of a hyaluronic acid-
antitumor
bioconjugate.
Bioconjug Chem. 1999 Sep-Oct;10(5):755-63.
PMID: 10502340 [PubMed - indexed for MEDLINE]
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The following message was posted to: PharmPK
dear simon,
maybe the following article helps: streiber et al. (2005) JPharm Sci.
Vol.
94 No.3: Methyl esters .... as drugs and prodrugs... (contains all
kinds of
stability measurements)
best regards,
Philip
Philip Lienau
Schering AG
Research Pharmacokinetics
Tel.: +49 - 30 - 468 - 18507
Fax: +49 - 30 - 468 - 12238
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Hi,
Thanks for the replies.
I am now designing a kinetic based screening (ranking) assay for
approx 50 compounds to look at parent compound inter conversion, due to
plasma esterases.
I intend to employ a 96-well plate format for triplicate samples over
four incubation time-points T=0, 30, 120 and 360 minutes.
I know that each time point for each compound should be performed on
the same plate (same day) with MeOH spikes as a reference standard ( I
will run parent compound linearity and precision stds), but surely it
is detrimental to the incubations to leave terminated (precipitated)
reactions in the well plate while awaiting the next time point ??
If so, the other option is to use separate plates (separate days due to
analytical restrictions) for each incubation time. Is the constructed
percentage pro-drug loss/ parent production ratio versus time profile
still valid this way? ie can I calculate relative decrease/increase in
pro/parent from T=0
values with confidence?
If not, how else would you go about it?
Your insight would be most appreciated.
Regards
Simon
Simon Clark M.Sc.
DMPK Specialist,
Analytical Pharmacology,
Antisoma Research Laboratories,
St Georges Hospital Medical School,
Cranmer Terrace,
London SW17 0QS.
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Hi Simon,
Why not use instead a larger tube, with enough volume to allow all your
scheduled sampling from the same tube. In such and experiment there
should be no issue with T=0 value. You can work on 3 parallel tubes if
you desire replicate results.
However, I will advise for the analysis of data within each tube and
comparing the rate constants at the end.
I hope this help,
radu
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The following message was posted to: PharmPK
Esterase can be added at different time intervals and all samples
extracted at the end, giving you time kinetic.
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Ok thanks,
So, if I was to use the homogenous incubation approach, ie three
wells/tubes and sampling at the times specified, can I get away with
taking 100ul aliquots for each timepoint from a 600ul mixture?
As there are 5 time points, removing 100ul at a time for sample prep,
in theory should not affect the kinetics, is this really true in
practice? I know the concentrations should remain relative. But I have
always thought that there is a discrepancy in removing such a large
proportion from the mixture.
My grasp of enzyme kinetics isn't amazing, but am I right in saying
that the rate of reaction is determined by the total protein/enzyme
present and although the substrate concentration remains the same, the
reaction will not proceed with the same rate constant
therefore not producing the same amount of product as that of the
previous (greater volume) incubation?
Thanks
Confused
Simon Clark M.Sc.
Analytical Pharmacology,
Antisoma Research Laboratories,
St Georges Hospital Medical School,
Cranmer Terrace,
London SW17 0QS.
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