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Dear all,
I have one querry regarding Peff calculation in SPIP model using rat
intstine where, Permeability Effective= Qx Ln(Cin/Cout)/2Pi RL, where
Cout is out let concentration from intestine.
Lets consider a drug is susceptible for intestinal metabolism and so
it will give lower Cout and finally will leads to give over estimated
Peff value.So how can we calculate Peff for such kind of drug.
Many thanks,
Rahul vats
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The following message was posted to: PharmPK
Rahul,
Single pass intestinal perfusion reflects only drug clearance from the
gastrointestinal tract, across the apical surface into the cytosol for
small
molecules undergoing passive diffusion.
It does not suggest the mechanism of clearance, viz. whether the drug is
degraded, metabolized, passes into lymph vessels, deposits into the
submucosa (which may act as a sink depending on the drug properties) or
sinked away into the mesenteric circulation, accumulated in organelles
and
so on.
The regional uptake rate constant from your cannulated or excised tissue
includes any unknown processes contributing to drug disappearance, thus
chemical and enzymatic stability incubation studies would need to be
performed to extract out or account for the metabolic portion of the
rate
constant. Have a look at a number of papers from Lennernas, who has
contributed significantly to this area.
Good luck
-Shawn
Shawn D. Spencer, Ph.D., R.Ph.
Assistant Professor of Biopharmaceutics
Florida A&M College of Pharmacy
Dyson Bldg., Rm 227
Tallahassee, FL 32307
shawn.spencer.at.famu.edu
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Dear Rahul,
This is the assumption that has been ruled out for a long time for
SPIP in the BCS Peff experiment and now people already started to get
concerned in this fact and the modified BCS called BDSCS
(Biopharmaceutics drug disposition classification system) is coming
into the picture which consider the regional GIT metabolism in
calculation of the Cout hence Peff.
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Dear Rahul,
To avoid such overestimation of Peff, many peoples do mesentric
canulation
and collect the blood samples. These blood samples further processed to
know the concentration of drug at different time point.
by doing this one can avoid overestimation of Peff.
hope this will hellp you.
regards
Dr. Amol A. Raje
Pharmacology Lab.
New Drug Discovery Department
Wockhardt Research Centre
D - 4 MIDC Area, Chikalthana
Aurangabad 431 210 M.S.
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Thanks sir
For explaining in such a nice manner. One thing what i wanted to know
as you mentioned only for passive diffusion. Will it be applicable for
molecules which is transported through Carriers or facilitated
diffusion?
Regards,
Rahul Vats
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Dear Rahul,
The permeability model you described assumes a linear behaving system at
steady state. The Peff is an apparent net parameter, so again, it
includes
MDR effects, unstirred layer effects, parallel carrier/passive
mechanisms...you name it... its in there.
You won't know the mechanisms of uptake unless you perturb the system
for
nonlinearity, as a carrier system is perfectly capable of behaving in a
linear fashion under observation. You can add some transporter
inhibitors
to the perfusate (compared to co-administration of some passive
diffusion
markers) if you suspect your drug has affinity for amino acid
transporters,
OCTs OATs etc, but I always like to say... let the structure drive the
experiment. There are enough in silico tools available to predict
membrane
transport with some degree of confidence... for small molecules.
Unless your study is purely academic, carrier effects seem not to be a
concern but for reasons to believe the Fabs will be "excessively" low or
variable. By excessively, I mean there are practical drawbacks.
All the best,
-Shawn
Shawn D. Spencer, Ph.D., R.Ph.
Assistant Professor of Biopharmaceutics
Florida A&M College of Pharmacy
Dyson Bldg., Rm 227
Tallahassee, FL 32307
shawn.spencer.aaa.famu.edu
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The following message was posted to: PharmPK
Dear Amol Sir,
AS you mentioned here the collection of blood at different time points
from mesentric vein to assure the Peff. But again i am fail to
understand the use of this blood concentration data in Peff
calculation. And further wanted to know that concentration measured in
mesentry will be in blood or in plasma matrix as in different matrix
concentration will be different?
Many thanks,
Rahul Vats
Novel Drug Discovery & Development
Lupin Ltd. (Research Park)
46A/47A, Village-Nande,
Taluka- Mulshi
Pune-411 042, India.
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The following message was posted to: PharmPK
Rahul,
The mesenteric concentration is not used to calculate Peff, and is not
an
indication of absorption. It is an indication of absorption minus
first pass
metabolism in the gut wall, minus drug retained in the gut wall, and
minus
drug that gets into the lymphatic system. There might be some other loss
mechanisms in some cases between the time the drug is absorbed and
when it
reaches the mesentery.
Two important things to remember:
(1) Peff is a measure of the rate of disappearance of drug from the
lumen.
That's why lumen concentrations, not mesentery concentrations, are
used in
its calculation. Peff is used to calculate rate of absorption.
(2) Absorption is simply getting out of the lumen. This could be by
entering
the apical bilayer of the enterocytes, by entering a transmembrane
protein,
or by getting into the paracellular pathway (probably beyond the most
apical
tight junction). Absorption takes place before gut wall metabolism, and
before drug moves into the lymphatic system.
One of the practices in the industry is to use in vitro cell culture
permeability experiments (Caco-2, MDCK, etc.) to try to predict Peff.
This
is commonly done by comparing the Papp from the cell culture
measurement for
a new compound with the Papp of one or more reference compounds with
known
Peff. That works pretty well most of the time.
The caveats are:
(1) the in vitro experiment measures only drug that managed to pass
all the
way through the monolayer, not the amount of drug that entered it on the
donor side,
(2) the in vitro monolayer cells are not the same cells as those in
different regions of the intestinal tract, so transporters and/or
enzymes,
tight junction gap dimensions, or other factors that differ between
the two
can produce significant differences in absorption and/or first pass
extraction,
(3) the conditions of the experiment are not the same as the
conditions in
vivo (e.g., drug concentrations, solvent chemistry, pH gradient).
Even Peff measured in humans is only valid for the perfused region and
under
the experimental conditions, so that in vivo absorption may not always
be
well-predicted using experimental human Peff. This occurs when the
absorption rate is much different in other regions of the intestinal
tract
for various reasons (e.g., transporter expression levels or surface/
volume
ratio) or when the conditions (e.g., pH, fluid volume) are significantly
different.
I hope this is helpful.
Best regards,
Walt Woltosz
Chairman & CEO
Simulations Plus, Inc. (NASDAQ: SLP)
42505 10th Street West
Lancaster, CA 93534-7059
U.S.A.
http://www.simulations-plus.com
E-mail: walt.at.simulations-plus.com
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Dear Professor,
Many thanks for the beautiful explanation.
Yes, Peff calculation is purely the linear estimates but the factors
governing it are fairly nolinear. And while employing the transporter
inhibitors also, one should take care of the regional expression of
the transporters that vary widely along the various GIT regions, MRP,
BCRP and P-Gp are to name few. Again, the fact of the structurally
driven experiments is very true but they are not exclusive untill
tested, though prediciton works more better for the OCTs OATs.
Unstirred layer effect is something difficult to mimic on the
experimentally acessible level which in silico perform better. At the
same time, GIT metabolism/degradation is one totally unrelated factor
with the aforementioned ones and rather easier to pinpoint (it can be
anallysed/quantitaed practically).
Amrit
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Dear Rahul,
As explained by Walt, mesentric concentrations are not used to calculate
Peff. It will just guide you, how much amount is entering into the
circulation. There are grossly two method of calculating Peff in situ
experiments.
One is static method explained by Doluisio and second where intestinal
segment is canulated along with mesentric or portal and jugular vein is
also canulated. Jugular vein is canulated for perfusing fresh whole rat
blood. Venous drainage of small intestinal loop can be collected via
single mesentric vein or portal vein and continually replaced with fresh
heparisnised whole rat blood which is constanly infused via jugular vein
from reservior. This "autoperfusion" method using whole fresh rat blood
ensures that homeostasis of the gut tissue is maintained.
For further understanding you can refer paper by Blanchard et al
(1989) J.
Pharm Sci. Vol. 79(5) pages : 411-414
Hope this will help .
regards
Dr. Amol A. Raje
Pharmacology Lab.
New Drug Discovery Department
Wockhardt Research Centre
D - 4 MIDC Area, Chikalthana
Aurangabad 431 210 M.S.
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Dear All,
If we consider this question about Peff, then will it be better to
carry out a set of experiments which will highlight the intestinal
metabolic stability, Pgp or other transporters susceptibility,
stability in the buffer in which the SPIP will be carried out.
Also various in vitro permeability models have their own limitations
which will highlight only about absorption aspects but not the above
mentioned aspects.For eg the expression of metabolic enzymes, Pgp in
MDR non-transfected MDCK, over tight junctions in CaCO cells etc.
Once contributions by these factors determined then one can have a
fair idea about the pretty accurate Peff.
Regards,
Devang P. Shah.
Senior Research Fellow (PhD Tech Fellow),
Dept of Pharmaceutical Sciences and Technology,
Pharmacology Lab II,
Institute of Chemical Technology,
N. P. Marg,
Matunga, Mumbai 400 019.
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