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I have4 subseries ofcompounds administered per os thatare allvery
quickly eliminated after 4-8h, with bioavailabilities ranging from 0.5
to 17% Fz and Cmax from50 to 500ng/ml at 10mg/kg. Biliary excretion is
believed because of the highest concentration of the compound as the
parent was found in bile (at the end of the study and not
glucuronidated but am't of glucuronides were not mesured)but not in
liver neither in plasma nor urine. Glucuronidation (on parent and
metabolites) was demonstratedin vitro fora numberof compounds.
However, some stuff are kind of weird: as example compound A has a
lower first pass metabolism in rat (30-40% met.) versus compound B
(50-55% met.), compound B gives much higher number of glucuronides than
compound A, solubility in buffer at pH 7.4 isbetter for compound B (50
microg versus 10 microg for Compound A), PPB is the same for both
10-12% fraction unbound. But in the end Compound B has a better
bioavailability 15% Fz versus 3.5% for compound A. Overall on the 4
sub-series there is no correlation for those cpds between any in vitro
parameters collected and the Fz obtained in the end. Excepted that all
Fz are very low and AUC desperately poor. A strong biliary excretion of
the parent itself is believed for those compounds that would
eventually have a good absorption and also be substrate of pgp-mediated
biliary excretion (no data). An other possibility would be a poor
absorptionbecause of the low plasma level from the start (first point
at 30min) and eventually more or less efficient enterohepatic
circulation that would make those compounds more or less
bioavailable.Very recently, I had a compound surprisingly from one of
this 4 sub-series with 50% Fz and pretty good AUC and Cmax; again the
in vitro parameters collected (Metabolism, PPB,....) are not better
than other compounds with poor Fz. Should I conclude that this compound
is not substrate of pgp-mediated biliary excretion or that it has a
more efficient active transport by intestin-pgp.
Do you know whether bile duct cannulated rat or mice are commercially
available from specific suppliers ? in order to check the biliary
excretion hypothesis ?other explanation possible for those compounds?
I would very much appreciate expertise from anyone.
Patrick Page
Dept. of Medicinal Chemistry
Serono Pharmaceutical Research Institute
Geneva (Switzerland)
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Patrick,
biliary cannulation is very simple in rats and
probably you can perform it yourself.
Sunil Bajad
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Bile duct cannulated rats are readily available from Charles River.
They instrument the rats with a shunt from bile duct to duodenum.
Simply cut the shunt and attach catheters for bile collection. The
rats will pass approximately 0.5 - 2ml/hour for the first many hours.
It is possible to then reconnect the shunt at the end of the experiment
for post 24 hour collection. You can also have blood collection or IV
administration catheters in the same model. They cost (depending on
the number of catheters) approximately $120-$180/each. They do good
surgery and the animals arrive in pretty good health. Good-luck.
Stanley Hollenbach
Senior Director, In Vivo Sciences
Portola Pharmaceuticals
Dear Patrick,Back to the Top
What is wrong with trying an acute bile duct cannulation experiment? It
is short term, so? You get an answer about what amount goes into bile
after po (or even iv, if you want to circumvent absorption issues to
answer this question about bile secretion). An other thing is: do the
acute bile duct and dose IV and collect intestinal luminal content,
then you will know about direct secretion into the gut. Whether this
will be Pgp related is an other issue.
What do you mean by no correlation between in vitro and in vivo
parameters? You mean in vitro (intrinsic clearance underpredicts what
you have found in vivo? E.g.). Also, I guess the max oral
bioavailability (defined as 1-hepatic extraction ratio) is not giving
you the correct prediction. Well, if your compound is relatively poorly
soluble, is a good pgp substrate, and a good cyp3a(4) substrate,
chances are you will already have a high first pass effect in the
intestine.
Maybe it helps a bit
Hans Smit, PhD
Drug Metabolism & Pharmacokinetics (PRBN-S)
70/121
F.Hoffmann-LaRoche Ltd AG
CH-4070, Basel Switzerland
Tel:+41-(0)61-688 7036
Fax:+41-(0)61-688 2908
johan_willem.smit.aaa.roche.com
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Dear Hans,
Nothing wrong in doing bile duct cannulated rat. I am trying to speed up
things in having those animals already from commercially available
sources
and Stanley recently provide me the information (thanks again). I should
mention that I am not a specialist of pharmacokinetics nor
phamacodynamics
as you guys, but just a medicinal chemist who try to make his best to
undestand issues with compounds for specific project, because of our
pharmacokineticians do not have really the time due to the number of
projects to propose eventually the most appropriated studies.
This said, your idea of doing acute bile duct, dose IV and collect
intestinal luminal content to determine the direct secretion into the
gut seems very interesting to me. But if you collect into the bile, how can
you still have compound in the lumen after iv administration ? something I
did not picked up here ? Also would you say that collecting in the lumen
will certainly affect drastically any possible enterohepatic circulation or does
the collection will affect it only partically and if so what proportion
of the enterohepatic circulation would you trap in collecting in the lumen?
Does collecting through a catheter in the lumen a tricky experiment to
perform ?
Also, I found in the literature that active transport mediated biliary
excretion is at least done through MDR3, MRP2 and MRP3, is it actually
limited to those transporters or is there any others ?
What I mean by no correlation in vitro vs in vivo is: there is no
parameters
in vitro collected to date (first pass metabolism in rat, PPB, solu at
various pH, permeability in PAMPA or Caco-2 (A->B/B->A), glucuronides
formation with or without NADPH) that can explained the ranking of the
compounds obtained in the in vivo PK rat studies p.os based on measured
Fz or overall plasma exposure to date. furthermore, only one single
compound very recently show very good plasma exposure and high Fz (50%) and
this again without having any better in vitro profile and even worse verus
bad Fz cpds (versus the parameters hereabove mentioned). That is why strong
biliary excretion is suspected for most of the previous compounds which is
something we did not manage so far.
Thanks for helping hands,
All the best
Pat
Patrick Page
Dept. of Medicinal Chemistry
Serono Pharmaceutical Research Institute
Geneva (Switzerland)
patrick.page.at.serono.com
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Hello Patrick,
I have definitely seen compounds whose clearance (and low
bioavailability)
are dominated by rapid biliary excretion of parent. I have been working
with radiolabeled versions of several analogues with differing metabolic
stabilities. In all cases the radiolabel was rapidly excreted in the
bile
(e.g. 80% of a 2.5 mg/kg dose in 0 - 4 h) and metabolic stability only
determined the proportion of parent present in the bile. I have to say
I
did not formally check to see if the rate limiting step was active
uptake
by the liver or active secretion in the bile but I actually suspect the
former. Caco-2 studies also showed significant efflux.
We have had very good experience with cannulated rats from Hilltop
(Scottdale, PA). I have no idea if they have a European supplier. We
use
animals with dual cannulae so we can perfuse them with taurocholate.
The
cannulae stay patent for at least 24 h, and usually 48 h or more.
I hope that this helps.
All the very best,
Bernard
Bernard Murray, Ph.D.
Senior Research Investigator
Drug Metabolism, PCS, PPD, GPRD, Abbott Laboratories, Chicago, USA
Bernard.Murray.-at-.abbott.com
Dear Patrick,Back to the Top
Dear Hans,
Nothing wrong in doing bile duct cannulated rat. I am trying to speed
up things in having those animals already from commercially available
sources
and Stanley recently provide me the information (thanks again). I
should mention that I am not a specialist of pharmacokinetics nor
pharmacodynamics
as you guys, but just a medicinal chemist who try to make his best to
understand issues with compounds for specific project, because of our
pharmacokineticians do not have really the time due to the number of
projects to propose eventually the most appropriated studies.
Inserted:
It's your money. An acute cannulation is not too difficult and quickly
done.
There is one other BUT that I have but this is a bit distracting from
your question I think. An operation in rats (or any other species) will
initiate an inflammatory response. I always find it a bit difficult to
interpret data from such experiments in light of the fact that proteins
involved in clearance of your compound be it transporters or phase1
and/or phase2 enzymes will be differentially regulated due this
inflammation response. I think one has to be a bit careful with what we
want to get out of such experiments. If your compound is a good
substrate for any protein (enzyme if you like) that will change in
expression level you might find some surprising things. I am not saying
this will happen, one just has to check carefully.
This said, your idea of doing acute bile duct, dose IV and collect
intestinal luminal content to determine the direct secretion into the gut
seems very interesting to me. But if you collect into the bile, how can
you still have compound in the lumen after iv administration ? something I
did not picked up here ? Also would you say that collecting in the lumen
will certainly affect drastically any possible enterohepatic circulation or
does the collection will affect it only partically and if so what proportion
of the enterohepatic circulation would you trap in collecting in the
lumen? Does collecting through a catheter in the lumen a tricky
experiment to perform ?
Inserted:
Anyway, the drug in the gut lumen will have to come from something
else if the bile is collected. Basically, the gut epithelia are pretty
good in secreting compounds that are taken up from the central
circulation into the gut lumen (which is in my view a function of
epithelia, this is what liver epithelial cells and renal proximal
tubular cells do). I would think the only reason why this seems
contradictory to our ways to go about is that normally drugs are dosed
orally. Hence one wouldn't SEE direct secretion. Will this influence
enterohepatic circulation. Well I would think so. To what extent
depends on the compound, local free drug concentration, among others.
Also, I found in the literature that active transport mediated biliary
excretion is at least done through MDR3, MRP2 and MRP3, is it actually
limited to those transporters or is there any others ?
There are others. Most likely you mean MDR1 rather then MDR3. Gets a
bit complicated looking at MRP's since they are not all localized in
the same domain, hence their roles may be different. Anyway, there is
BCRP which is pretty highly expressed in the epithelia of the gut.
What I mean by no correlation in vitro vs. in vivo is: there is no
Parameters in vitro collected to date (first pass metabolism in rat,
PPB, solu at various pH, permeability in PAMPA or Caco-2 (A->B/B->A),
glucuronides formation with or without NADPH) that can explained the
ranking of the compounds obtained in the in vivo PK rat studies p.os
based on measured
Fz or overall plasma exposure to date. furthermore, only one single
Compound very recently show very good plasma exposure and high Fz
(50%) and this again without having any better in vitro profile and
even worse verus
bad Fz cpds (versus the parameters hereabove mentioned). That is why
strong Biliary excretion is suspected for most of the previous
compounds which is something
we did not manage so far.
Regards,
Hans
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Dear Johan,
The information on inflammatory response due to cannulation is new to
me.
Do you have any references from literature? Or is this just common
knowledge for people how know the practical aspects of this kind of
cannulation. Do you know whether this response is seen in all kind of
species or is it restricted to rats/rodents/???.
From a regulatory point of view I do not have the impression that the
effects of such an inflammatory response would or could be so
substantial
that they would affect the overall conclusions on metabolism as a
registration dossier contains quite extensive data on metabolism.
Kind regards, Adrienne
AJAM Sips, PhD head department Method and Model development
National Institute of Public Health & the Environment
Center for Substances and Integrated Risk Assessment, U339
P.O. Box 1
3720 Bilthoven
The Netherlands
tel. + 31 30 274 2043
fax. + 31 30 274 4475
e-mail: adrienne.sips.aaa.rivm.nl
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Dear Adrienne,
I think if such operation is performed one will get inflammatory
responses simply because of the nature of biological systems. How severe
this turns out maybe dependent on how well these operations are
performed, condition of the rat maybe even rat strain etc.
What we have seen for compounds we tested in naive rats and bile duct
cannulated rats is that PK profiles are very different (following of
course same dose same route and keeping things the same as needed for,
and of course these animals where NOT bile salt depleted because such
experiment would be useless from my point of view). So that tells me
something is the matter, what ever this is: differences in metabolism
and/or transport functions?
The inflammatory responses I have read mostly about have often been
related to sepsis. Bottom line for all science data out there is that IL
1beta, IL6 and TNFalpha are able to 'deregulate' transporters and
metabolic enzymes expressed in liver.
Regards,
Hans
We have been using a chronically catheterized rat model forBack to the Top
pharmacokinetic studies in which the animals are not studied for 4 days
after surgery to specifically avoid the adverse effects of 'protocol
stress'. Studies performed in rodents within ~3 days of surgery and
anesthesia likely have compromised physiological relevance.
This citation may be of interest.
Am J Physiol Heart Circ Physiol 276: H671-H678, 1999.
Ray Galinsky
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Members of the discussion of the group,
We, too, have seen altered PK in bile-duct cannulated rats compared
with naive rats. With one compound, we saw increased AUC in bile duct
cannulated rats relative to naive rats. When we used bile-duct
cannulated rats with the bile reintroduced into the duodenum we got the
same results as the cannulated rats that did not have the bile
reintroduced, pointing to the surgical stress as the root cause. The
compound had <1% biliary excretion. My opinion, based on >20 years of
doing these studies is that these studies deliver yes/no answers. Is
biliary excretion important or not? One should not take the kinetics
seriously except in a very broad sense. If the half-life or AUC in rat
drops 10 fold, that's important (for compounds that are extensively
recirculated, I have seen this dramatic a change). If it changes 50%,
not much is going on. As a rule, I would not even collect blood (this
is additional stress on already stressed animals) or very limited
sampling, but would measure the urinary and fecal excretion. The loss
of bile salts is a significant stress in a rat, so I recommend infusion
into the duodenum of bile from donor rats or a solution of bile salts
in saline on any study lasting 24 hours or more. Another factor is the
length of recovery time after surgery, the longer the recovery time the
better, but the risk of lack of patency in the cannulas goes up.
Although the limitations of these experiments must be considered, I am
a strong advocate of these studies as early as possible in development.
IMHO, the sooner you know about biliary secretion the better. If the
drug still appears in the GI tract after bile duct cannulation, then
you have luminal secretion, also valuable to know.
Dale
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