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Dear all,
I have performed IV pharmacokinetic study . But as soon as the drug
reaches the systemic circulation it converted to metabolite and
complete drug disappeared within 30 second. Even the drug having
plasma stability which was performed for 24 Hour. Metabolic
stability was also performed with liver microsomes for 1 hour, drug
got metabolited upto 75% not completely. But after liver microsome
metabolism, metabolite obtained at different retention time from the
peak obtained in pharmacokinetis study with same chromatographic
method. No peak obtained at the retention time of metabolite peak in
pharmacokinetic study even after 3 Hour. This compound has limited
solubility in aquous solution. I want to know how fast the metabolic
conversion rate can be and if there is any effect of formulation how
can it effect. My formulation contain PEG with Tween 80. Could you
please recomend some suitable vehicles to use? Is there any effect of
tween 80 or PEG with some molecule.
I will be very pleased if i get the answer of this questions.
Thank you very much!
Gautam
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Gautam,
If true, this is extremely rapid metabolism and you should be able to
track it down. You might repeat stability with whole blood.
My first impression is that you are having a sample stability problem
(What was withdrawn from the rat is not what you are measuring
several hours or days later) or an analytical problem and not true
metabolism. Depending on your compound you may have to add esterase
inhibitors, clean up the samples with liquid:liquid or solid phase
extraction, use a refrigerated autosampler, adjust pH. You should
also make sure your anticoagulant is the same for your PK study and
the plasma you are using for your standard curve. You only mention
retention time so I am assuming you are using UV detection and would
intemperate a peak with shifted retention time as a different compound.
Good Luck. These sorts of problems are what keep the job interesting.
Mike
Michael Cameron, Ph.D.
Scripps Florida
Department of Drug Discovery
DMPK Group Leader
5353 Parkside Dr RF-2
Jupiter, FL 33458
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The following message was posted to: PharmPK
Dear Gautam,
Have you tried the drug stability in whole blood and what about the
tissue distribution of the drug? Is the drug rapidly distributed and
sitting in the tissues? Is the drug stable in the formulation? Hope you
find the solution,
Cheers,
Jagannath
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The following message was posted to: PharmPK
Gautam,
Have you ever noticed significant stability difference
in different species in your in vitro studies? Did you
do metabolite stability studies in hepatocyte in
different species?
Xiaodong
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The following message was posted to: PharmPK
Dear all:
we have had a similar experience and we finally found that strong red
cells
binding (and also extensive metabolism) was one of the reasons for
such low
plasma levels. If you don't have an analytical method for whole
blood, try
spiking whole blood with a known amount of your analyte, incubate 5
to 10
minutes at 37oC, centrifuge and analise plama sample. If
concentration is
much lower than expected, think in blood cell binding or blood cells
related metabolism.
Alvaro Ganza
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if in vitro studies showed different metabolites profiles from in
vivo, I would put more credits on in vivo data and try more matrix
like hepatocytes or cofactor-supplemented microsomes in in vitro
studies. You mentioned you tested plasma stability, how about whole
blood?
I usually saw some quatitative effects of formulation on metabolic
conversion in vivo probably by affecting the absorption, but I doubt
there influence in qualitative aspect.
Hong Lu
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Hi Gautam
1.) My first question would be was the i.v injection given properly?
2.) On which animal specie was this data generated?
3.) You mentioned that you had performed stability studies in plasma.
my next Q would be if you had conducted studies in whole blood. There
could possibilities of drug distributing into the RBC's. You could
perform RBC uptake studies to find that out.
4.) You also mentioned that there were qualitative differences in the
metabolite profiles in in vitro exp's compared to that in in vivo.
What were the other means of structure elucidation (e.g. LC/MS/MS,
PDA etc) apart from HPLC retention times to show that the metabolite
obtained invitro was not the same invivo.
I am asking these questions because you said that the retention times
were different in both samples using same chromatographic method. How
big was the difference in Rt? Sometimes small changes in column
performance, solvent composition, environment temp, mobile phase flow
rates (leakages in HPLC system) etc could influence Retention times.
PEG is fine but try to use the minimum quantity that allows to
solubilize the drug.
An alternate route to check metabolites would be I.P route (in
animals). If i remember one could administer suspenions by this route
so then you dont have to worry for solubilization (Just a thought).
Best Wishes.
Manish Issar, Ph.D
Sandoz
4700 Sandoz Drive
Wilson, NC-27893
USA
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Hello Gautam, I would first look into the solubility of the compound
and ask if it is precipitating out somewhere during the course of the
study e.g. could be precipitating out during analysis due to mobile
phase composition (have you double checked if you are getting
appropriate standard curves?) or as soon as it reaches the systemic
circulation due to change in formulation composition.
Alternatively, the drug might be having a high volume of distribution
(is the drug lipophillic, or is it known from its structures that it
could be a potential substrate for some influx transporter?)
Regarding your formulation, what % PEG are you using ? A few % change
in PEG can affect solubility by a large extent.
Also what do you know about the drug molecules' non specific binding
(to apparatus).
Lastly, what is your study design, IV bolus or infusion and at what
dose level? You have performed stability in plasma and I would
recommend doing the same in whole blood.
If possible, also give the drug orally to a couple of rats and see if
you get any peaks or not?
I am sorry that I am not able to give you an exact suggestion, but
you have to trouble shoot and first find out what are NOT the causes
for this erratic behaviour, then you can narrow down your search and
find out what are the causes.
Indranil Bhattacharya
Principal Scientist
DMPK
GlaxoSmithKline
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Gautam,
As you mentioned that metabolite appearing in PK study has different
RT than metabolism study. Additionally your compound is stable in
plasma for 24 hours. This essentially means that compound is unstable
in whole blood.
I experienced the same problem while analyzing the benzofuroxone
derivatives, there was ring opening within 30 seconds and formation
of amino-nitro derivative.
I would suggest to go for whole blood stability first.
Regards,
Jignesh Kotecha
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The following message was posted to: PharmPK
Hi Gautam,
It is good idea to obtain the stability information of
the NCE in biological matrices before you proceed for
analysis and characterization. A standard format
include stability in whole blood for enzymatic
degradation, partitioning into RBC etc.
If it is enzymatic degradation you can try inhibitors
(you would find in the old queries at this site). If
it is degradation or simple conversion then you have
to be really quick in separating plasma (as your drug
is stable in plasma). Collecting blood in chilled
tubes (heparin or EDTA) would also be helpful.
Hope this helps.
Jagdish
Jagdish Jaiswal, PhD
ADME Pharmacologist
Auckland Cancer Research Centre
University of Auckland
Auckland
New Zealand
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