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Dear all,
we are facing a unique feature for the class of
oxazolidinone compounds, in which we were conducting
the stability studies of drug with blood. we incubated
the drug in blood at 37 C and aliquot of blood was
taken at different time intervals and analysed the
drug levels. In this study, we found with time, the
aliquot of blood taken from 37 C and when removed an
aliquot of blood to room tempt(22C), the blood gets
clotted.wheras At 37 C, the incubated blood is fine.
1.Is the drug has influence on thrombin or fibrin.
2. Is the protein binding of the drug plays a major
role
3. What is the relevance of the room temperature and
37 C. can we assume that the drug behaviour at body
tempt(37C) will be ok. This behaviour(22C vs 37C) will
cause toxic effect in the body in some disease
conditions.
Can someone give some suggestions on this behaviour.
with regards
sivakumar
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Hai Shivakumar
This type of problem we noticed long back for two
reasons
1) If you use the whole blood without any anti
coagulant (only for serum)
2) If you add concentrated anti oxidants.
And i hope you will get more information if you talk
to any Biochemist.
with regards
nageshwar rao thudi
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Dear Sivakumar,
Do oxazolidinones have effect on thrombin!?
What is the volume of the aliquot you take for each time point?
Is this an increase in blood viscosity or clotting?
Do you cap the tube during incubation?
How do you spike the drug into the blood, which solvent?
The probability of a drug behaving differently at temperature as close
as
these may not be significant but, that is the last question?
have you tried incubation of aliquots in individual tubes and drawing
one
out at a time to avoid errors, this is a better approach than drawing
aliqouts at different points from a single tube.
Hope this helps,
Jagannath
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dear rao,
we used heparinised blood and there is anticoagulant
which is present(heparin).We added drug to the whole
blood and there is no problem at 37 C, but we see the
problem when we take the incubated blood from 37 C to
room temperature(22c).
Actually there is slight increase in the viscosity of
the blood when we removed the blood from 37 C to room
temperature. Another unique observation is even on
centrifugation, we could not get plasma. I am very
surprised by this behaviour of the drug on incubation
with blood. could anybody throw some ideas on this.
what could be the property of drug which could cause
this unique behaviour.
regards
sivakumar
--
dear Jagannath,
Volume of aliquot which we used is 200ul each time
from the initial volume of 7ml blood. But the after an
hour, the aliquot of 200ul blood was removed from 37c
and it really became viscous. Even on centrifugation
of this blood, we could not get plasma. I donot
understand this. You rightly pointed out that there is
only increase in the viscosity but not a really clot
formation.The solvent used is acetonitrile which is
less than 5% in the final volume of blood. At 37C, the
drug incubated with blood is as usual, only on taking
the blood to room temperature, we see this behaviour
of increase in viscosity and on centrifugation also,
plasma could not be obtained.
Even similar pattern is obtained on incubation of drug
in blood at 37 C when we did as a single aliquot.
I am not able to understand the unique aspect, even
not able to get plasma also in these samples.
What is the reason for increasing in viscosity at room
temperature.
Why we are not getting plasma.
regards
sivakumar
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Are you using the whole blood or plasma? At the
temperatures mentioned here (22 and 37), no difference
in the clotting behavior is expected.
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Hai sivakumar
One more question is have you tried the total procedue
without adding the drug (ony with blood + solvent you
are using to spike the drug) to find out the problem
is due to drug or something else like temp. effect,
solvent, RPM set etc
with regards
tn rao.
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I incubated the drug in blood at 37 C and after
different time intervals, an aliquot of blood was
taken and analysed. so the unique observation, the
blood when taken out from 37c and brought to room
temperature, it becomes a viscous and even on
centrifugation, we could not obtain plasma.
But control sample, the blood without addition of drug
at 37 C, there is no problem and clear plasma is
obtained. I want to understand what is the intersting
feature of the drug which made the above observations.
can somebody tell the reasons for not getting plasma
also and temperature sensitive behaviour.
regards
sivakumar
--
dear nageshwar,
The control blood with similar conditions without the
addition of drug is fine. we did not face any problem.
we got plasma and did not observe this jelly or
increase in viscosity problem.
what could be property of drug which makes this
happen.
regards
sivakumar
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Hai Siva kumar
It is good,now you know the precipitation is due to
drug and some thing else interaction. I suggest one
more thing to try is you repeat the process in K3 EDTA
blood or plain blood.
I hope this will help you out
nageshwar
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Dear Siva Kumar,
Have you used the same vehicle, I mean everything same, but for the
drug in
the control?
What is the anticoagulant you had used?
Is your compound a calcium salt?
Is there any interaction between the compound and the anticoagulant that
makes the anticoagulant inactive?
Hope this helps,
Jagannath Kota
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Siva,
Did you try to use spiking Linezolid in the same solvent as that of
your compound. try that and see how your compound is structurally
different from LNZ. That might give you some clue. Try spiking your
compound in serum or plasma and see what happens. Why you want to use
only blood and not serum ?
Sandeep
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dear sandeep,
We spiked the drug in plasma and there is no problem
in recovery or stability. The drug spiked in blood and
incubated at 37c, when aliquot of blood taken to room
tempt and we see the increase in viscosity and blood
donot separate into plasma even on centrifugation. I
donot understand the strange behaviour. One another
intersting aspect is drug added to blood and kept at
37 C is ok, when analysed as blood is fine.
regards
savithiri shivakumar
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