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Dear Colleagues:
I was wondering if someone has come across a case where a compound
shows good absorption in the monkey (as determined by the total plasma
radioactivity AUC ratio following oral and IV administration of
radiolabeled compound) despite poor intrinsic permeability in the
intestinal absorption model, Caco-2.
The possible explanation we came up with was (a) permeability could be
underestimated as compound is very lipophilic and practically
insoluble, and (b) compound could be transported by active/carrier
mediated processes in the monkey and these transported could be absent
in the Caco-2 model (closer scrutiny of the structure, however
suggests passive transcellular mechanism).
Now I have two specific questions (with some subquestions) :
1. If (a) is true, can someone suggest an approach to estimate true
permeability (cosolvents and solubilizers like DMSO, cyclodextrin,
0.4% methyl cellulose + 0.5% tween80 have already been tried
but the compound seems to be insoluble because even the transwell
filter itself is proving to be rate limiting)? What about the
effect of unstirred water layer?
2. Is total radioactivity ratio (oral/IV) a right approach to
calculate fraction absorbed in animals (or humans)? Under what
circumstances this approach would not work (such as may be
insoluble compounds)? What will be the best approach to calculate
fraction absorbed (follow renal recovery, but if compound is
excreted in bile!)?
I would highly appreciate your thoughts and comments.
Gopal
--
Gopal Krishna, Ph.D.
Drug Metabolism and Pharmacokinetics, Schering-Plough
Email: Gopal.Krishna.-at-.spcorp.com, Phone: 908-298-6564 (work)
Editor and maintainer : Pharmacokinetics/Biopharmaceutics home page
http://griffin.vcu.edu/~gkrishna/PK/pk.html
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Dear Gopal,
I suggest that there might at least be one other explanation to your
problem. If your substance is highly lipophilic it might be dissolved
within emulsified fat micelles in the lumen thereby providing a rapid
equlibrium between the tiny fraction of drug that is dissolved ( and that
can be absorbed) and undissolved drug. The absorbed drug can then be
transported into the circulation along the fatty acid pathway- contained in
triacylglycerol chylomicrons that is secreted into the lymph system and
then mixed into the circulation when lymph drain into Vena Cava.
As might be seen thismis also a way of hepatic "escape".
Nils Ove Hoem, Ph.D
Assoc. Professor
Dept. of Pharmacology, School of Pharmacy,
University of Oslo,
POB 1068 Blindern
N-0316 Oslo
NORWAY
Phone: +47 22 856561
Fax: +47 22 857511
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Dear Gopal Krishna,
I think you should add a possible explanation c) with:
c) The drug was disintegrated in the GI tract after administration to
monkeys and the product of disintegration is good absorbed. With this you
will find a good absorption determined by the total radioactivity and you
can find a poor intrinsic permeability in the intestinal absorption model.
To clarify the discrepancy you should separate the radioactivity in plasma
samples into to compounds (by TLC) and compare the radioactivity given by
the unchanged drug.
Willi Cawello
Clinical Pharmacology, Schwarz Pharma AG
D40789 Monheim, Germany
EMail Cawello.-at-.SchwarzPharma.com
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Dear Dr. Krishna,
I think measuring plasma conc. based on total radioactivity may cause
problem if one does not know what he is measuring, parent drug or
metabolite(s). Indeed it is possible you have over estimated oral
bioavil. due to presystmic metabolism in the GI tract. If metabolites
formed in the gut still radioactive and enter systemic circulation,
total radioactivity measured in blood or plasma will be total
radioactivity associated with both parent molecule and metabolite(s).
Therefore, the AUC after po administration will be over estimated.
Regards
Majid
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It seems that I need to provide some more information so that a more
meaningful discussion can be made regarding my original queries (Poor
permeability but high absorption).
Willi's point is right. What if the compound is disintegrating? We
also though about that but the bioavailability (AUC oral/AUC iv) from
the LC/MS/MS analysis of the parent compound in plasma itself was
moderate to high.
Majid rightly pointed out that radioactivity measurements can cause a
problem if one does not know what he is measuring (parent compound or
metabolite). What if one does know that he/she is measuring both and
the position of the radiolabel is such that the radiolabel is going to
be either on the parent drug or on the metabolite(s)? Can we still get
a reasonable estimate of the total percent absorbed (total radioactive
AUC oral/ total radioactive AUC iv). Again, under what circumstances
(such as physicochemical properties of compounds or physiological
factors not related to radioactivity) this approach won't work?
Nils offered the most plausible explanation based on physicochemical
properties and physiological approach. Thinking on somewhat similar
lines, we also tried experiments in the Caco-2 (human intestinal
absorption model) in the presence of the simulated intestinal fluid
and still permeability did not increase (permeability was less than
the permeability of a poorly absorbed compound, mannitol, which was
included as an internal/leak marker). However, fatty acid pathway, as
suggested by Nils, could be absent in the Caco-2 model, thereby,
resulting in the underestimation of permeability (and poor solubility
will further contribute to the underestimation of permeability).
My thanks to all of you who responded to my original email.
Regards,
Gopal
--
Gopal Krishna, Ph.D.
Drug Metabolism and Pharmacokinetics, Schering-Plough Email:
Gopal.Krishna.-a-.spcorp.com, Phone: 908-298-6564 (work)
Editor and maintainer : Pharmacokinetics/Biopharmaceutics home page
http://griffin.vcu.edu/~gkrishna/PK/pk.html
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Dear Gopal
There is a possible explanation for the second question you address. The ratio
oral/IV AUC is a correct way to determine the F value only if you meassure
the same
compound when given iv or oral. It can happen that when you give the drug,
after
absorption through the intestinal wall, it is metabolized and the
metabolite retains
the radioactivity. Therefore, when you determine the radioactivity you may be
calculating the radioactivity due to the metabolite rather that the drug
radioactivity, and having a high oral/iv AUC ratio that can be mistaken by
a high F
value. A closer look at its metabolism and the enzymes responsible for its
biotransformation both at the gut wall and the liver would be very valuable.
You also mention that is very lipophilic and almost insoluble. I like to
point out
the case of cyclosporine A where its low bioavailability is not only due to
its low
solubility but also to a high gut metabolism (mainly by CYP3A isoenzymes) and
P-glycoprotein counter-trasport back to the intestinal lumen. These two
phenomena may
be happening with your drug.
I can provide you with two references at this moment that can give you some
ideas:
Benet LZ, Wu CY, Herbert MF, et al. Intestinal drug metabolism and
antitransport
processes: potential paradigm shift in oral drug delivery. J. Controlled
Release.
1996, 39:139-143
Gomes DY, Wacher VJ, et al. Effects of ketokonazole on the intestinal
metabolism and
bioavailability of cyclosporine. Clin. Pharmacol. Ther. 1995, 58:15-19
I hope this information gives you some help. If you need something else
contact me.
See you
Ignacio Segarra, Ph.D.
Postdoctorate
Dpt. of Biopharmaceutical Sciences
University of California at San Francisco
513 Parnassus Ave. (room U-66)
San Francisco, CA 94143-0446
e-mail:nacho.at.itsa.ucsf.edu
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> > 2. Is total radioactivity ratio (oral/IV) a right approach to
> > calculate fraction absorbed in animals (or humans)? Under what
> > circumstances this approach would not work (such as may be
> > insoluble compounds)?
If you use the traditional oral/IV AUC ratio you should recognize that
this is only a valid method of estimating the extent of bioavailability
when you are willing to assume that systemic clearance is first-order.
If your drug has capacity limited elimination then AUC also becomes
dependent on the rate of drug input as well as extent.
--
Nick Holford, Dept Pharmacology & Clinical Pharmacology
University of Auckland, Private Bag 92019, Auckland, New Zealand
email:n.holford.-at-.auckland.ac.nz tel:+64(9)373-7599x6730 fax:373-7556
http://www.phm.auckland.ac.nz/Staff/NHolford/nholford.html
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Reply (or question) to Nick Holford,
I agree that AUC becomes dependent on rate of drug input as well as extent
when elimination
is limited, but I would be interested to know which examples you had in
mind and why
elimination is limited in their cases. Since by definition almost, input
must exceed
elimination to produce any AUC, there must be some kind of minimum
elimination rate below
which this significantly affects AUC calculations and I cannot remember
seeing any
discussion of that before.
Andrew Sutton. Guildford Clinical Pharmacology.
ASutton.-a-.gcpl.co.uk
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The term "capacity limited" is also known as
"saturable","Michaelis-Menten","concentration dependent" elimination.
The most obvious example is ethanol. Unfortunately some authors have
drawn erroneous conclusions about the first pass metabolism of ethanol
by using AUC to calculate bioavailability. It is possible to have an
oral AUC greater than an IV AUC if the oral dose input rate is
sufficiently fast in relation to the IV input rate.
e.g.
First order:
dC/dt = (Ratein - CL*C)/V
Capacity Limited:
dC/dt = (Ratein - Vmax*C/(C+Km))/V
The maximum elimination rate (the "capacity") is Vmax. Note that the
actual elimination rate is driven by C at any realistic value of C in
relation to Km. The term "zero-order" elimination is not a reasonable
description of this kind of elimination model.
--
Nick Holford, Dept Pharmacology & Clinical Pharmacology
University of Auckland, Private Bag 92019, Auckland, New Zealand
email:n.holford.aaa.auckland.ac.nz tel:+64(9)373-7599x6730 fax:373-7556
http://www.phm.auckland.ac.nz/Staff/NHolford/nholford.html
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Nick Holford,
Mpharmacokinetics) also thouight that alcohol was an obvious example but no
therapetic agents come to mind.....
Andrew Sutton
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In response to Nick Holford's comments on capacity limited
metabolism and absorption of ethanol (see below).
It's true that using AUCs can yield erroneous interpretations
regarding the extent of absorption (F) for drugs which follow
"capacity limited" metabolism, as was used in some of the first
papers describing the First Pass Metabolism (FPM) of alcohol.
Clearly, F=AUCpo/AUCiv assumes First-Order kinetics, whereas
alcohol approximates Michaelis-Menten elimination kinetics.
But a method has been devised and used to evaluate the
absorption of alcohol (1) which takes into account its MM kinetics.
The solution is to use an "integration method" to calculate the
quantity of alcohol (Q) reaching the systemic circulation by the
IV and PO routes, and then define F=Qpo/Qiv (2).
Q = Vd * Int(t=0 to infinity) Vmax * Ct/(Ct+Km) * dt
While the Quantity (Q) could be calculated as either an absolute
amount (mg) or a relative dose (mg/Kg), the latter is used
because mg/Kg is a typical unit of dosage in alcohol studies.
As a partial validation of the values of Vd, Vmax and Km used to
make the calculation, the Qiv should equal the dose administered.
A continuous Qt. can be calculated by adding a factor for the
Quantity of alcohol in the body at any time point "t."
Qt. = Vd * Ct. + Vd * Int(t=0 to t.) Vmax * Ct/(Ct+Km) * dt
This latter equation is similar to an "absorption plot" used by
Wagner and his colleagues to describe the "efficiency" of
alcohol absorption (3).
(1) e.g. Roine et al., Alcoholism: Clin Exptl Res 15:734 (1991)
and Lim et al., Alcoholism: Clin Exptl Res 17:1337 (1993)
(2) This is analogous to F=CL*AUCpo/CL*AUCiv for
First-order drugs. Also, since alcohol is completely
absorbed from the gastrointestinal tract, FPM=Qiv-Qpo.
(3) e.g. Lin et al., Res Comm in Chemical Path Pharmacol
13:713 (1976)
Tom Gentry
Bethesda, MD
email: tgentry.at.willco.niaaa.nih.gov
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Phenytoin is the classical therapeutic agent with capacity limited
elimination.
--
Nick Holford, Dept Pharmacology & Clinical Pharmacology
University of Auckland, Private Bag 92019, Auckland, New Zealand
email:n.holford.-at-.auckland.ac.nz tel:+64(9)373-7599x6730 fax:373-7556
http://www.phm.auckland.ac.nz/Staff/NHolford/nholford.html
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)