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Dear All,
Greetings!!!
I have performed PK study in SD Rats by intravenous route. To my
surprize, the plasma level concentration was increasing upto few time
points. Is it possible to get this type of pk profile upon iv
adminsitration. My formulation was 10% DMSO, 10% Ethanol (Acidified),
10% PEG and the rest is water. Formulation became turbid upon addition
of water but it was administered without any problem.
Help is needed with possible explaination/reasons. It would be a great
help if you could guide me about the maximum conc. of DMSO and Ethanol
which may be used for IV formulation.
--
Thanks and Best Regards,
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Hello-
A simple answer may be that you have extravasated the dose and what
you see is your measured compound absorbing from the extravascular
tissue surrounding the injection site (or where the cannula is
implanted). This is a reasonably common occurrence with direct
injections into rat veins.
Lane Brunner
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The following message was posted to: PharmPK
Dear Wahaj,
IV injection of a turbid formulation (suspension) is not, in general,
indicated, unless it was voluntarily designed as such (nano-suspension).
As some material is not entirely dissolved it may take some time for
part of the compound to dilute/dissolve again in blood, thus causing
plasma levels to truly increase with time.
Another explanation to your awkward PK results may be that it is an
analytical artefact, rather than reality, due to matrix effect of the
formulation on the analysis. PEG may interfere with ionisation during
LC-MS/MS causing the plasma levels to be under-evaluated, I had
experience with ion suppression of up to 90% loss in signal through
matrix interference. As PEG is itself eliminated from the blood-stream
the effect is very strong at early time-points and non influential at
longer time-points, causing the bell shaped IV profile (resembling
that of an oral dose).
Patrice Larger
Pharmacokinetics, Metabolism & Dynamics
Translational Sciences & Pharmacokinetics Dpt
ROTTAPHARM | MADAUS
patrice.larger.at.rottapharm.com
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The first thing I would check is your bioanalytical method, as it
looks like you may be getting ion suppression from PEG, with earlier
samples suppressed a lot more than the later ones, as PEG gets cleared
from the body. This is very common and easy to check - take a couple
of the early iv samples, split each into two equal parts, then spike
one part with a known amount of your analyte standard and analyze. If
the difference between the spiked and non-spiked samples is correct,
suppression is not a problem. If not - you know where your problem is...
Hope this helps,
Andrew Volosov
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Dear Group,
I am not answering this question, but seeking suggestions on
bioanalytical method development in such a scenario.
It is suggested that analyte suppression may be expected in early time
points. Does it mean, whenever there is PEG used in formulation, one
need to check for signal suppression effect due to PEG? If there is a
suppression effect observed, are there any processing methods
specifically to remove PEG?
Regards,
Vinayak
Vinayak Nadiger
Manager, Bioanalytical Chemistry
11 Biopolis Way, Helios #08-05
Singapore 138667
E Mail: vnadiger.-at-.combinatorx.com
Website:www.combinatorx.com
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Vinayak,
The answer is yes, absolutely, you need to verify that ion suppression
is not an issue, and not only when PEG is used in the formulation (but
with PEG - definitely). Many other things can potentially cause
suppression. The problem is that the extent of suppression may - and
will - vary from sample to sample. Trying to remove PEG is hardly a
viable option, as it would be difficult to develop a PEG-specific
extraction (especially considering the overwhelming amount of PEG in
the sample, compared to the analyte) , and even if you did, you would
not be sure about other possible interfering components in the sample.
Playing with chromatography does not really help either, because PEG
would often overload the column and elute as a huge long "wave".
Therefore, the easiest way to go is to compensate for suppression,
rather than trying to get rid of it. Stable isotope-labeled internal
standard will do it very nicely 99% of the time. If you do not have
the labeled IS, you have to measure the extent of suppression directly
in your samples, and then tweak your method, if necessary. Here's how:
1. Take a few actual samples (three should be enough), preferably the
early iv and 0.5 - 1h oral samples (the idea is to pick the samples
most likely to be suppressed, when the concentration of PEG is the
highest).
2. Split each sample in two equal parts, then spike one part with a
known amount of your analyte. The amount should be large enough to be
easily measurable accurately.
3. Analyze these samples and look for the correct difference between
the spiked and non-spiked samples. If the difference is equal to what
you spiked - you are all set. If not, you have to play with the LC
method and keep re-analyzing your spiked and non-spiked samples until
you are able to measure the correct difference. One trick that works
very well is using an internal standard that is structurally related
to the analyte, and force them to co-elute as closely as possible.
When you do that, in most cases, the IS will get suppressed to the
same extent as the analyte, and your measured result will be correct.
The main point of the above approach is that you prepare samples that
allow you to have a direct probe that tells you whether your method is
accurate, and if it is not, you have a reference point you can use to
modify and re-test your method quickly and easily. You cannot assume
that there is no suppression - you have to measure it to prove its
absence.
Hope this helps.
Andrew Volosov
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The following message was posted to: PharmPK
Dear Wahaj,
I agree with the views expressed by other group members that abnormal
PK profile following i.v. administration could be due to ion
suppressing effect of PEG in your formulation.
We also faced similar problem with one of our molecule.
We were initially using protein precipitation method for analyzing the
same earlier but later changed to Liquid-liquid extraction method and
eluted the PEG peak. This helped resolve the problem to some extent.
I don't have the exact details of the bioanalytical method.
Hope this might of some help to you.
Regards
Tausif Ahmed, Ph.D.
Principal Scientist, DMPK,
Sai Advantium Pharma Ltd., Pune, India
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The following message was posted to: PharmPK
Hi Andrew,
Thanks for the detailed discussion. I do underastand choosing a
labelled IS is the best case scenario. In most of discovery research,
we tend to use a generic IS , or at the best structural analog which
may elute as close as possible. My concern is, method will generally
be validated using blank plasma, which will not contain PEG. Thus,
validation parameters are not reflecting the actual situation. How do
regulators react for such observations?
Regards,
Vinayak
Vinayak Nadiger
Manager , Bioanalytical Chemistry
11 Biopolis Way, Helios #08-05
Singapore 138667
E Mail: vnadiger.aaa.combinatorx.com
Website:www.combinatorx.com
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Dear Wahaj,
Do you see any trend in internal standard peak area during study
sample analysis? If the peak area of internal standard remains
constant and it is co-eluting with analyte then chances of ion
suppression is less or in rare situation, there could be specific
effect (ion suppression) on analyte only. If area is less in earlier
time points one can confirm the ion suppression by diluting the PK
samples (e.g. 10 times) with control plasma and reanalysis. If the
repeat conc. is comparable then look for other reasons.
regards,
Jignesh
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Hi Vinayak,
The FDA guidance says that "...appropriate steps should be taken to
ensure the lack of matrix effects, ...especially if the nature of the
matrix changes from the matrix used for method validation". Naturally,
they do not specify the steps that should be taken. Therefore, most
often people simply test their method with blank plasma from a number
of different sources, which is clearly not enough. And you are right -
strictly speaking, it is impossible to perform a meaningful validation
without specifically addressing what may be happening in the actual
samples. Fortunately, by the time validated methods are required
people usually will have a labeled IS synthesized. However, in
discovery all bets are off and you have to be particularly diligent.
If for some reason you need to validate a method without a labeled IS,
you have to specifically demonstrate that the actual samples are
measured correctly, not just the QCs prepared from blank plasma.
Regards,
Andrew
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Hi Andrew, Vinayak,
In discovery environment it is difficult to have labelled IS. In one
of our study where we observed ion suppression during initial
timepoints, we asked for placebo administered plasma samples and check
analytes' recovery and matrix effect against normal plasma
calibration. This helped us to resolve the problem.
regards,
Jignesh
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The following message was posted to: PharmPK
Dear Wahaj,
As you mentioned that your formualtion became turbid upon dilution
with water is an indication of precipitation. Upon iv administration,
the dose might have precipitated due to rapid dilution of your
cosolvents resulting in depot formation of your drug at the injection
site (solubility issue). Such type of formulations might exhibit an
increase in measured concentrations at later time points. You should
consider altering your formulation for intravenous dose.
Chandra Durairaj,
Univ of Colorado Denver
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I would try to stay away from using DMSO IV if you can at all. Using
very aggressive formulations can give you erroneous drug hopes for the
compound. Also you are more likely to cause hemolytic responses that
can and will interfere with your bioanalysis.
--
Sanjeev Thohan, PhD
SARx Consulting
SARxconsult.aaa.Gmail.com
http://www.linkedin.com/in/sanjeevthohan
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