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The following message was posted to: PharmPK
Dear colleagues,
for our drug screening we routinely use serial blood sampling from the
tail vein to establish plasma PK in CD-1 nude mice. Typically, we take
4 to 5 small samples over 2 to 3 hrs. Blood is collected in heparinized
hematocrit capillary tubes and spun to collect the plasma. Recently, we
noticed differences in the plasma concentration-time curve when
comparing single (terminal) bleeds (from the retro-orbital sinus under
halothane anaesthesia) obtained from multiple mice (one sample per time
point per mouse) with serial tail vein bleeds: initially, the plasma
concentration was lower, then there was a cross-over point followed by
later times at which the plasma concentrations were higher in the
samples obtained from the tail vein. There was no difference between ip
and iv administration when sampling from the retro-orbital sinus (in
heparinized microtainers; see below) at any time point. In all cases
plasma PK were mono-exponential over the measured period (but with
different half-lives, of course). Samples from the retro-orbital sinus
were collected using either hematocrit tubes or larger heparinized
microtainers. When taking three samples in rapid succession from the
same mouse (performed in triplicate) at an early time point the
measured plasma concentrations were as follows (in order of sampling):
from the tail vein (hematocrit tube; lowest conc.), retro-orbital sinus
(hematocrit tube; intermediate conc.), and retro-orbital sinus
(microtainer; highest conc.). So, it appears that plasma is not a
homogenous compartment (retro-orbital sinus vs. tail vein), or at least
not as homogenous as we had hoped. In addition, the observed
differences may also have something to do with the sampling method
(hematocrit tube vs. microtainer). My question is whether other people
have had similar experiences or aware of similar findings (literature
refs.)? Any useful comments & suggestions are appreciated.
TIA
Frederik Pruijn
Experimental Oncology Group
Auckland Cancer Society Research Centre
Faculty of Medical and Health Sciences
The University of Auckland
New Zealand
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Frederik:
Did you notice any difference in the color of the plasma? We've
observed
that there is more hemolysis in tail vein bleeds because of the physical
manipulations of the tail required to make the vein visible. The plasma
is
thus "pinker" than blood drawn through a catheter. This would matter if
the compound is inside the blood cells. This is simple enough to check.
Take 2 blood samples (e.g. from a cardiac puncture) and spin one into
plasma. Freeze the other blood sample, thaw, and then spin the blood and
pull off the supernatant. Compare the concentration of drug in both
samples.
Best Wishes,
Candice Kissinger
BASi
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The following message was posted to: PharmPK
Dear Frederic,
There is a nice study by Balani et al., 2004 comparing
the serial and terminal blood sampling in mice. They
stated the PK of antipyrine administered iv to mice
followed by serial and terminal sampling yielded
similar results. However, if you look at Fig 1 in the
pub, you will see that up to 2 h the
concentration-time data mirror each other but after 2
h they start deviating with serial method giving
higher concentrations. Please see Drug Metabolism and
Disposition Vol 32, No. 10, 1092-1095 for details.
Rostam
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The following message was posted to: PharmPK
Dear Frederik,
The rodent tail is a rather poorly perfused appendage. Therefore, when
you sample blood from the tail vein, you are not really sampling the
central compartment, but a peripheral compartment, and your
pharmacokinetic results will be affected accordingly. We abandoned the
tail vein for pharmacokinetics long ago. (you may want to try serial
sampling from the saphenous vein if you do not want to do serial retro
orbitals in mice - there have been a few publications on this method).
Sincerely,
Peter Rix
Ligand Pharmaceuticals
San Diego, CA
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The following message was posted to: PharmPK
Dear Candice,
thanks for your comments. I have thought about hemolysis playing a
role. However, there is no difference in colour between plasma obtained
from a sample from the tail vein compared to one from the retro-orbital
sinus (and neither are pink). The only manipulations we apply to take a
sample from the tail vein is to use a heat lamp; we don't actually
'massage' the tail (apparently, some people do this). The other
argument against hemolysis is the fact that IMO this should cause more
variability in the samples and this not what we have observed: the
variability is very similar with tail vein vs. retro-orbital sinus
(unless the level of hemolysis is similar in ALL samples at ALL time
points, which I find hard to believe). The findings with the drug are
mimicked by its main (more polar) metabolite. But, as you said, it is
simple enough to check whether the drug (and its main metabolite) are
in blood cells and this is an interesting piece of information per se.
Kind regards,
Frederik Pruijn
--
Frederik B. Pruijn PhD MSc (Senior Research Fellow)
Experimental Oncology Group
Auckland Cancer Society Research Centre
Faculty of Medical and Health Sciences
The University of Auckland
Private Bag 92019
Auckland
New Zealand
Phone: +64-9-3737 599 x86939 or x86090
Fax: +64-9-3737 571
E-mail: f.pruijn.-at-.auckland.ac.nz
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The following message was posted to: PharmPK
Dear Rostam,
I checked out the article, thanks.
I quote: "The tail vein bleeding frequently led to a degree of
hemolysis after the initial couple of time points; as anticipated, this
led to the overestimation of plasma concentrations (data not shown)
because of the possible partitioning of AP [FP: antipyrine] into red
blood cells. Sampling from cannulated mice did not cause hemolysis"
This confirms what Candice Kissinger was saying.
Kind regards,
Frederik Pruijn
--
Frederik B. Pruijn PhD MSc (Senior Research Fellow)
Experimental Oncology Group
Auckland Cancer Society Research Centre
Faculty of Medical and Health Sciences
The University of Auckland
Private Bag 92019
Auckland
New Zealand
Phone: +64-9-3737 599 x86939 or x86090
Fax: +64-9-3737 571
E-mail: f.pruijn.aaa.auckland.ac.nz
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The following message was posted to: PharmPK
Dear Peter,
do you happen to have any lit. refs? We have applied for ethical
approval to do serial sampling from the saphenous vein.
What you're saying makes a lot of sense but it is also a little
worrying as we (and many others) use the tail to do iv administrations.
With some drugs we have indeed observed the tail taking on the colour
of the drug solution (more so than the rest of the mouse, if you can
see & say this) after iv injection into the tail vein and there were
suggestions of poor tail perfusion. At the time we thought this was
(mainly) caused by local vascular damage caused by the drugs in
question (formulated in saline). Suffice to say that we could not
sample from the tail vein (on the other side of the tail) as the
measured concentrations were much higher and very variable compared to
terminal bleeds. Interestingly, after a couple of hours the
concentrations in tail vein samples approached those in terminal
samples but this was well and truly in the elimination phase when
concentrations had decreased considerably. In addition, more or less
warming of the tail using a heat lamp did seem to make a difference
(but we didn't really connect this with the other pieces of
information). Taking this altogether with the other comments posted to
PharmPK I now subscribe to the idea of the tail vein representing a
tissue rather than the central compartment.
I have to say that there is surprisingly little info on this in the
literature and the little there is is not that easy to find......
Perhaps we should write this up for publication; any suggestions for a
journal anybody?
Kind regards,
Frederik Pruijn
--
Frederik B. Pruijn PhD MSc (Senior Research Fellow)
Experimental Oncology Group
Auckland Cancer Society Research Centre
Faculty of Medical and Health Sciences
The University of Auckland
Private Bag 92019
Auckland
New Zealand
Phone: +64-9-3737 599 x86939 or x86090
Fax: +64-9-3737 571
E-mail: f.pruijn.at.auckland.ac.nz
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The following message was posted to: PharmPK
Dear Frederik,
Here is a reference for serial saphenous collection:
Hem A, Smith AJ, Solberg P.
Saphenous vein puncture for blood sampling of the mouse, rat, hamster,
gerbil, guinea pig, ferret and mink.
Lab Anim. 1998 Oct;32(4):364-8.
The method involves collecting small samples (< 100 uL) into a
"microvette" (Sarstedt). Repeat samples are collected by basically
removing the scab and drawing blood into the microvette by capillary
action. The microvettes can then be centrifuged for plasma or serum
isolation. Because of the small sample size, you will need a fairly
sensitive bioanalytical method - LC-MS/MS, ELISA, etc. If you keep your
collection vol below 50 uL, you should be able to take at least 6
samples from a 25 g mouse without exceeding 15% of total blood volume.
The maximum depends on your institution's IACUC guidelines. You may
also want to administer some saline (s.c. or i.p.) once or twice during
the study.
Hope this helps.
Peter Rix
Ligand Pharmaceuticals, Inc.
San Diego, CA
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The following message was posted to: PharmPK
Dear Koen,
very useful information and thanks for the reference.
In one of my earlier posts I did mention that we have applied for
ethical approval to do serial bleeds on mice via the saphenous vein.
However, a colleague of mine has already tried the reverse, which you
describe for rats, and this seemed to work well on mice too.
Nevertheless, if plasma from the tail vein indeed represents a
peripheral compartment, as others have argued, we would still have a
problem, regardless of the route of administration.
Kind regards,
Frederik Pruijn
--
Frederik B. Pruijn PhD MSc (Senior Research Fellow)
Experimental Oncology Group
Auckland Cancer Society Research Centre
Faculty of Medical and Health Sciences
The University of Auckland
Private Bag 92019
Auckland
New Zealand
Phone: +64-9-3737 599 x86939 or x86090
Fax: +64-9-3737 571
E-mail: f.pruijn.-at-.auckland.ac.nz
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The following message was posted to: PharmPK
Dear All
What is the best way to dose either dexamethasone or rifampicin into
mice ? Dosage form , dose ... any idea ?
What is the best way to get blood sample from mice , what is the
maximum
volume ?
Mohammad shawaqfeh
Clinical pharmacy
University of Iowa
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The following message was posted to: PharmPK
Hi Frederik:
There is another way of collecting serial blood samples from mice:
automated blood sampling. We've been doing this in rats and guinea pigs
since 1999, but two years ago we adapted the technique for mice. You
can
view a paper on this at
http://www.bioanalytical.com/info/poster/pdf/CBK-09.pdf and on page 10,
you
can see time-concentration curves from 4 mice for carbamazepine and its
epoxide metabolite. Each mouse generated 8 samples, all of which were
collected from a freely-moving animal. At the same time, we collected
behavioral information and mouse urine since the animal was in a
movement-responsive metabolic cage. We used the same automated blood
sampler that we use for other rodents, but with a modification to the
software to adapt to the total blood volume of a mouse, and using a
no-blood-waste software method which allows us to draw small samples and
waste no blood in the process. The limiting factor in implementing
this
technology is the surgery to implant a catheter in the mouse. For IV
dosing, we need a 2nd catheter, and it looks like the combination of a
jugular catheter (dosing) and carotid catheter (blood collection) is the
best. For oral dosing, we've adapted the gastric catheter we use in
rats
(for automated oral dosing) so we don't have to handle the mouse at all
for
either IV or oral dosing. We're exchanging ideas with mouse vendors to
see
whether it will be possible for them to provide precannulated mice -- as
they do for rats. For those PharmPK members who already cannulate mice
(there were references to this among replies to your query), they've
already accomplished the hardest part of this technique.
Candice
Candice B. Kissinger
Senior V.P. Research
BASi -- Bioanalytical Systems Inc.
2701 Kent Avenue
Purdue Research Park
West Lafayette IN 47906-1382 USA
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The following message was posted to: PharmPK
Dear Candice,
very impressive stuff! Perhaps not quite suitable for routine PK
screening during drug discovery/early drug development stages. My
former colleagues in Amsterdam were quite involved in radio-telemetry
in small lab-animals and your poster somehow reminds me of their work.
Kind regards,
Frederik Pruijn
--
Frederik B. Pruijn PhD MSc (Senior Research Fellow)
Experimental Oncology Group
Auckland Cancer Society Research Centre
Faculty of Medical and Health Sciences
The University of Auckland
Private Bag 92019
Auckland
New Zealand
Phone: +64-9-3737 599 x86939 or x86090
Fax: +64-9-3737 571
E-mail: f.pruijn.at.auckland.ac.nz
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The following message was posted to: PharmPK
Hello Frederick:
You bring up an issue that I focus on frequently. I often run
first-in-animal PK studies for discovery companies which may not have
access to a vivarium and which also have very limited supply of
compound.
It seems to me that we should try to get as much out of each PK study as
possible. Serial blood sampling in a single animal certainly makes sense
from the perspective of the amount of material available for dosing.
Why
dose 5 mice to get one PK curve when you can dose 1 mouse and get the
entire curve without interanimal differences? More importantly, why not
also get urine, behavior, body temperature, or tissue dialysates
representing free drug concentrations from the same animal? That animal
serves as its own control. Correlations of physiology with PK are
powerful. Telemetry is is a valuable technique..but it doesn't let you
collect biofluids for PK.
I would agree that this wouldn't fit into a PK screen that involved
hundreds of compounds per week. It would generate an enormous amount of
data. However, if the in vitro screens are generating focused targets,
this sort of approach can provide early indicators or more than PK
alone.
Best Wishes,
Candice
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The following message was posted to: PharmPK
To all:
I totally concur with the comments of Candice Kissinger. They make an
excellent point that some of the old techniques are no longer effective
and/or desirable. As Candice states: "Correlations of physiology with
PK are powerful". The key issue of her method, to study single animals
in a longitudinal study, rather than pool several animals for single
time points, is both very desirable and indeed necessary to obtain
meaningful results. We had investigated that issue many years ago (see
Noninvasive Estimation of Bound and Mobile Platinum Compounds in the
Kidney Using a Radiopharmacokinetic Model. R. R. Brechner, D. Z.
D'Argenio, R. Dahalan and W. Wolf, J. Pharm. Sci., 53, 873-877, 1986),
where we documented that pooling of animals obscures interindividual
differences, especially critical in those steps involving physiological
processes - renal or hepatic elimination, etc.
There was a cartoon in the older editions of Remington, where you saw
two bullet holes symmetrically located on each side of a duck that was
swimming contently. The legend below read: "On the average, the duck is
dead".
In addition, and while the sampling of accessible body fluids (blood,
urine, etc) is clearly useful for many drugs, most of the time it is
not meaningful for drugs acting on tumors and a number of other
pathological sites. PK data must be obtained from such target tissues,
which is still possible of the drug can be measured using noninvasive
imaging - either nuclear, NMR (MRI/MRS) or optical.
--
Professor Walter Wolf, Ph.D. President, Correlative Imaging Council,
Society of Nuclear Medicine
Distinguished Professor of Pharmaceutical Sciences
Director, Pharmacokinetic Imaging Program
Department of Pharmaceutical Sciences, School of Pharmacy
University of Southern California 1985 Zonal Ave., Los Angeles, CA
90089-9121
E-Mail: wwolfw.aaa.usc.edu
Telephone: 323-442-1405
Fax: 323-442-9804
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The following message was posted to: PharmPK
Dear Prof Wolf
I find a little bit confusing this statement in on of
your e mails ( and I would appreciate if you can
explain it to me on detail):
> In addition, and while the sampling of accessible
> body fluids (blood,
> urine, etc) is clearly useful for many drugs, most
> of the time it is
> not meaningful for drugs acting on tumors and a
> number of other
> pathological sites. PK data must be obtained from
> such target tissues,
> which is still possible of the drug can be measured
> using noninvasive
> imaging - either nuclear, NMR (MRI/MRS) or optical.
Do you mean tissue/tumor concentration and thus drug
pk in tissue/tumour has nothing to do with plasma
concentrations?
in that case BE requirements for drugs acting on
tumours should not be based on plasma levels (and
AUc)???
Does it mean is not posible to model the drug pk in
tumor tissue from the plasma levels?
(i that case I have ben reading hundreds of papers of
people trying to fool me...)
thanks in advance
Marival Bermejo.
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The following message was posted to: PharmPK
Marival:
See my answers below:
> Do you mean tissue/tumor concentration and thus drug
> pk in tissue/tumour has nothing to do with plasma
> concentrations?
> in that case BE requirements for drugs acting on
> tumours should not be based on plasma levels (and
> AUc)???
Yes, in most, if not all the anticancer drugs acting against solid
tumors, plasma levels have no direct relationship with drug
effectiveness/response. The explanation for that is twofold:
1. Perfusion of solid tumors varies significantly, so much so that in
patients with multiple metastases different masses are likely to exhibit
significant differences in perfusion, and hence, significant differences
in how much of a drug will reach each tumor mass.
2. Because solid tumors are not homogeneous, they will have different
activities in how anticancer drugs are transported into tumor cells.
This will be due to a combination of what happens in the interstitial
fluid space and what happens at the level of the cell membrane, and its
expression of influx and efflux transporters.
> Does it mean is not posible to model the drug pk in
> tumor tissue from the plasma levels?
> (i that case I have ben reading hundreds of papers of
> people trying to fool me...)
The short answer is yes. Plasma levels are only relevant for what
happens in plasma, and NOT what happens in tissues. The reason that
plasma levels are more relevant for non-cancer drugs is because those
target tissues are much more functionally homogeneous and predictable.
See for example "Tumor based pharmacokinetics of drugs has greater
significance for anticancer drugs than does blood-based
pharmacokinetics. Walter Wolf and Cary A. Presant, Clinical Pharmacology
and Therapeutics 76:508, 2004".
--
Professor Walter Wolf, Ph.D.
President, Correlative Imaging Council, Society of Nuclear Medicine
Distinguished Professor of Pharmaceutical Sciences
Director, Pharmacokinetic Imaging Program
Department of Pharmaceutical Sciences, School of Pharmacy
University of Southern California
1985 Zonal Ave., Los Angeles, CA 90089-9121
E-Mail: wwolfw.aaa.usc.edu
Telephone: 323-442-1405
Fax: 323-442-9804
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The following message was posted to: PharmPK
Walt
thanks for your detailed explanation.
> Yes, in most, if not all the anticancer drugs acting
> against solid tumors, plasma levels have no direct
relationship with drug effectiveness/response. The
explanation for that is twofold:
I followed and understand the explanation but from my
point of view a "no direct relationship" is different
from a "non-existing" relationship.
Even if complex, or consisting on multiple step or in
nonlinear processes that relationship exists.
So I still think pk concentration is relevant for
effectiveness/response in tumour tissues.
On the other hand I could agree with you that your
methodologies can be of help to solve the difficulties
in modelling those complex relationships.
> The short answer is yes. Plasma levels are only
> relevant for what
> happens in plasma, and NOT what happens in tissues.
> The reason that
> plasma levels are more relevant for non-cancer drugs
> is because those
> target tissues are much more functionally
> homogeneous and predictable.
The non-homogeneity of the target tissue in tumours
just makes the sytem more difficult to model in
mathematical terms...it does not means that the
diffusion/transport laws do not work anymore.
checking the previous discussions about this point on
the list I can see that we are not going to agree
easily. I can see the value of imaging methods for
evaluating levels on target tissues..but possibly I am
not going to see the irrelevance of PK levels for most
drugs including cancer drugs.
Two different formulations F1 and F2 of cancer drug A
producing the same plasma PK profile, from my point of
view, will at the end produce the same tissue levels
in tissue X1, X2, X3....that will drive to response
Y1, Y2, Y3..in both cases unless
diffusion/transport/enzyme reaction laws work
differently with F1 and F2. If the formulation
components do not reach the plasma and what plasma
"see" is just drug A...I still do not see why we are
going to obtain different effectiveness/response
coming from the same plasma profile.
many thanks for the reference.
marival
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The following message was posted to: PharmPK
Dear Marival,
A agree with your post.
Assuming the source of drug for a tissue is from the plasma (instead of
direct injection, topical application, by direct contact through
inhalation for airways, etc.), then there MUST be a relationship between
plasma concentrations and tissue concentrations. Complex transport into
the tissue or complex pharmacodynamic effects may require an indirect
model, perhaps one that has not yet been discovered. But as you noted,
the fact that the model is unknown does not mean it doesn't exist.
Walt Woltosz
Chairman & CEO
Simulations Plus, Inc. (AMEX: SLP)
1220 W. Avenue J
Lancaster, CA 93534-2902
U.S.A.
http://www.simulations-plus.com
Phone: (661) 723-7723
FAX: (661) 723-5524
E-mail: walt.-at-.simulations-plus.com
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The following message was posted to: PharmPK
Marival:
Thank you for this very interesting discussion. Because I am now
teaching a course in Pharmacokinetic Imaging, I will share this
discussion and use it as a discussion exercise, including the
development of equations that may make this qualitative discussion a
little more quantitative.
There are however two issues that I need to address, because without
spelling those out we are continuing a series of different
understandings of what we are talking about. The first issue deals with
your comments on the relation of plasma PK levels and tissue levels,
and the second with the relation of such plasma levels to
effectiveness/response.
In a systems approach, there are 3 phases that need to be considered
when studying the flux of drugs in a living organism, each of which is
controlled by different determinants.
1. In the transport phase we estimate the rate and the amount of the
drug from its site of administration to its target site(s), while also
considering the competing processes to non-target tisssues and
elimination organs.
2. In the uptake phase we estimate the rate and the amount of the drug
that can be incorporated into the cellular space at the target site(s).
In solid tumors we need to consider both the role of the interstitial
fluid space (IFS) as well as that of the transport into and out of the
tumor cells.
3. In the metabolic phase we estimate the rate and the amount of the
drug that is converted to its anabolites and catabolites, and hence how
much of the active form(s) of the drug will be present at the site(s)
where it will exercise its therapeutic action.
We may also need to consider, in parallel to these pharmacokinetic
aspects, the toxicokinetic aspects and their role in
effectiveness/response.
That is why I will believe that your comment: "I still do not see why
we are going to obtain different effectiveness/response coming from the
same plasma profile" is answered by the fact that plasma profiles are
primarily relevant in the transport phase, but not in the uptake or the
metabolic phases. And effectiveness and response are a function of the
complete system, not solely of what happens at the first stage.
Obviously, poor drug availability in the transport phase will be the
rate limiting step, but even with good drug availability in plasma, if
not enough/inadequate rate occurs in the uptake and metabolic phases,
the effectiveness of the drug still needs that the rate/amounts at the
these two later phases be adequate.
One analogy I use in my classes and presentations is that of the two
countries where people are starving. In both countries we bring into
the airport/harbor all the food they need. But in country X, where we
do not have good roads or sufficient vehicles to transport the food,
people will continue starving, whereas in country Y, good roads and
sufficient transportation provide an effective answer to the problem.
Focusing only on one aspect of the system is inadequate in the case of
anticancer drugs. Effectiveness/ response requires suitable and
sufficient rate processes in all 3 phases.
--
Professor Walter Wolf, Ph.D. President, Correlative Imaging Council,
Society of Nuclear Medicine
Distinguished Professor of Pharmaceutical Sciences
Director, Pharmacokinetic Imaging Program
Department of Pharmaceutical Sciences, School of Pharmacy
University of Southern California 1985 Zonal Ave., Los Angeles, CA
90089-9121
E-Mail: wwolfw.-a-.usc.edu
Telephone: 323-442-1405
Fax: 323-442-9804
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The following message was posted to: PharmPK
Walt
wonderful! as a professor I can not resist to
participate in a teaching exercise:
> In a systems approach, there are 3 phases that need
> to be considered ....> 1. In the transport phase 2.
In the uptake phase > 3. In the metabolic phase we
estimate the rate and
up to this point there is no disagreement between
yours and my point of view
the fact that
> plasma profiles are
> primarily relevant in the transport phase, but not
> in the uptake or the
> metabolic phases.
but if you are talking of consecutive processes,
linked by some mathematical equation (more or less
complex) the last steps still are a consequence of the
plasma levels.
> the effectiveness of the drug still needs that the
> rate/amounts at the
> these two later phases be adequate.
I do not disagree with that. But you still have not
answered my question about why profile level A will
produce effct X and exactly the same profile level A
is not going to produce effect X.
(and I am not talking about hysteresis loops...)
>
> One analogy I use in my classes and presentations is
> that of the two ....
I fully agree with tha analogy..than means for me that
plasma concentrations can lead to effect in one tissue
and be no effective in a different tissue with
different composition/transporters/flux rates etce...
up to this point we are again in fully agreement.
But I want you to put again the discussion in the
"Comparison issue"
> Effectiveness/ response requires
> suitable and
> sufficient rate processes in all 3 phases.
yes, but my point is still that in those three phases
if you start in tha same strating point you will end
in the same end-point.
Could you show me experimental data comparing (I
repeat comparing) two equal plasma time profiles
leading to different levels in one tissue for
instance?
of course in different tissues I could obtain
different levels and eventually the effects/response
could be different..but I still think we could model
all these different tissues profiles from the plasma
level as a reasonable sampling site.
marival
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The following message was posted to: PharmPK
Dear Dr. Wolf
In your response to Marival you suggest that ?tissue/tumor
concentration and
thus drug pk in tissue/tumor has nothing to do with plasma
concentrations?,
and that ?solid tumors are not homogeneous?. You are stating further
that
?Plasma levels are only relevant for what happens in plasma, and NOT
what
happens in tissues. The reason that plasma levels are more relevant for
non-cancer drugs is because those target tissues are much more
functionally
homogeneous and predictable.?
My comment:
Plasma concentrations ARE relevant to that what happens in tissues.
However,
there is no predictable time/concentration relationship between plasma
levels
and tissue levels, especially TARGET tissue levels, due to differences
in
ADME. Target tissue levels are usually different from non-target tissue
levels. The two must not be confused and should be distinguished.
Yes, tumor tissues are not homogeneous; but neither are non-tumor
tissues.
Tumor and non-tumor tissues all are heterogeneous in composition and
function, with considerable variations. Non-tumor tissues may be more
complex, probably most are, than tumor tissues.
Evidence indicates that among different target-cell populations in vivo
specific drug-receptor binding, retention and kinetics may vary
significantly. Unfortunately, despite this, the determination of target
cell
uptake and kinetics is not required for drug approval and therefore not
usually done. Common ?expedient? approaches instead use whole organs or
chunks of organs with mixed populations. To be able to identify in vivo
target cell populations and related kinetics, microscopic resolution
would be
required in most cases.
From our studies of drug localization with tissue and cellular
resolution,
evidence indicates that drug levels differ between plasma and target
tissues
and that drug-receptor binding and kinetics may also vary considerably
among
different target tissues.
In the same tumor even, target cell populations can vary in uptake and
retention of drug.
To resolve such questions further, high resolution in vivo tissue data
are
essential and should be included in studies of drug effects. Certain
questions cannot be answered without it. I have discussed this in detail
based on evidence (see articles below).
Walter E. Stumpf
http://www.unc.edu/~stumpfwe
Stumpf WE (2005). Drug localization and targeting with receptor
microscopic
autoradiography. Journal of Pharmacological and Toxicological Methods
51(1):25-40.
Koike N, Endo K, Kubodera N, Kumaki K, Ikeda K, Ogata E, Stumpf WE
(1999)
In vivo nuclear uptake of a vitamin D analog (OCT) in different tumor
cell
populations of FA-6 cancer xenograft in nude mice by receptor
autoradiography.
Anticancer Res. (1999)Nov-Dec;19(6B):4955-8.
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The following message was posted to: PharmPK
Marival:
Thank you. We seem to be closing the gap, and I appreciate seeing how
we seem to agree on so many points.
Let me address here the "Comparison issue". I agree fully that starting
from the same point and all conditions being equal we obviously should
end at the same point. And that is probably the case in many, if not
most non-tumor tissues.
The problem with solid tumors is that, even when they have the same
origin (e.g., breast, colorectal, etc), they are not the same. As one
example, in a patient where we were able to measure separately the
tumoral half-life of 5-FU using 19F-MRS in 2 separate metastatic
lesions in the liver, one of the lesions had an elimination half-life
of 5-FU of 29.7 min, whereas the other tumor mass had an elimination
half-life of 4.1 min. The first lesion trapped 5-FU and was responsive,
the second lesion did not and the patient's disease progressed.
What will be interesting will be, after we develop a series of models
and set up the equations, we will have a testable set of hypotheses
which will can be analyzed with either existing data, or with new data
acquired specifically for testing these hypotheses.
--
Professor Walter Wolf, Ph.D. President, Correlative Imaging Council,
Society of Nuclear Medicine
Distinguished Professor of Pharmaceutical Sciences
Director, Pharmacokinetic Imaging Program
Department of Pharmaceutical Sciences, School of Pharmacy
University of Southern California 1985 Zonal Ave., Los Angeles, CA
90089-9121
E-Mail: wwolfw.-a-.usc.edu
Telephone: 323-442-1405
Fax: 323-442-9804
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The following message was posted to: PharmPK
Walt
> We seem to be closing the gap,...
> I agree> fully that starting
> from the same point and all conditions being equal
> we obviously should
> end at the same point. And that is probably the case
> in many, if not
> most non-tumor tissues.
gap almost closed.
this would be the same in tumor tissues.
I am not talking about the same end point in different
tissues. I agree with you in that part.
but starting point A will lead to end point 1 in tumor
1, end pont 2 in tumor 2, and so on...and if the start
is the same the different end points will be similar
(between ocassions not similar among them, that seems
to be your main point whith which I agree.)
so even if the starting point (plasma) could be less
relevant for response/effectivenes prediction UNLESS
we develope the adequate model for linking both
variables, it is useful for comparison purposes..
best wishes
marival
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The following message was posted to: PharmPK
Dear All
In my opinion there are two important questions in discussion.
1.- If the plasma levels have relationship with drug
effectiveness/response
2.- If the plasma levels are more relevant for cancer or non-cancer
drugs
considering the diffecences in the perfusion and the heterogeneity of
target tissues.
I'm in agreement with Marival and Walter Stumpf that plasma
concentrations
are relevant to that what happens in tissues, and that drug kinetics may
also vary considerably among different target tissues. In the practice
the
prediction of drug concentrations in target tissues depends on the PK
information in plasma and the behaviour of drug in specific tissues that
can be modelled using plasma levels information and relevant information
as tissue/plasma or tumour/plasma ratio related with the distribution
in
specific tissues. (Noe DA, Kumor KM. Drug kinetics in low-flux (small)
anatomic compartments. J Pharm Sci.72(6):718-9.1983.)
Certainly, the heterogeneity of tumour tissues implies alternatively the
use of more complex pharmacokinetics model as spatial models for the
prediction of tissue levels and response instead of conventional models
as
compartmental or classic physiologic models more appropiated for
relatively homogeneous (non-tumour) target tissues. However these
complex
models also needs the plasma levels information for the prediction of
drug tumour levels.
Ref:
Banerjee RK, Sung C, Bungay PM, Dedrick RL, van Osdol WW. Antibody
penetration into a spherical prevascular tumor nodule embedded in normal
tissue. Ann Biomed Eng. 30(6):828-39. 2002.
Lanao, JM, Fraile, MA. Drug tissue distribution: study methods and
therapeutic implications. Curr. Pharm. Des. In press. 2005.
Prof. Jose M. Lanao
Dpt. Pharmacy and Pharmaceutical Technology
Faculty of Pharmacy
University of Salamanca
Spain
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The following message was posted to: PharmPK
Dear Dr Stumpf:
> Plasma concentrations ARE relevant to that what
> happens in tissues. > However,
> there is no predictable time/concentration
> relationship between plasma
> levels
> and tissue levels, especially TARGET tissue levels,
> due to differences
> in
> ADME.
I find again this statement confusing and misleading.
Target-tissue concentrations may be different from
nonTarget tissue concentrations due to differences in
disposition processes. ok...and?
BUT there is a relationship (as complex as you wish)
between plama profiles and leves in ANY tissue. (and
thus finally with effect)
> Evidence indicates that among different target-cell
> populations in vivo
> specific drug-receptor binding, retention and
> kinetics may vary
> significantly.
Ok but for me, that means, that in different target
cell populations you have different cascades of events
linking the drug in plasma and the drug in the
receptor...that is all...
all these events can be describe/modelled in
mathemathical terms (of course with a set of
reasonable assumptions) using the
transport/reaction/diffusion laws..driven by the
corresponding concentration gradients...and including
if you need time delay terms....
this transport/reaction/diffusion events work today
the same way they work yesterday (including stochastic
variations).
I understand that both of you are pointing out the
interest of you experimental work and your
measurements of tissue levels.
I have never deny the interest of it.
But I felt compelled to answer Walt message because I
find misleading the way you put into words things,
implying the lack of relationship between plasma
levels and tissue levels and effect.
For a while now, we have been using bioequivalence
studies as surrogate for therepeutic equivalence, as
the main assumption is that site of action
concentrations are related (with all the complexities
you want..)to the plasma concentrations.
This assumption has worked so far and up to now nobody
has demonstrated that two similar plasma profiles lead
to different therapeutic effect...
but now that you have the means/methodologies for
meassuring tissue levels maybe you would be so kind of
demonstrating to all of us that two equivalent plasma
profiles will lead to completely different
distribution of drug in the body (in the thousand
compartment you wish to differentiate).(and I am not
referring to the fact that the concentrations in each
target, non target tissues would be different..)
we do not need even to put us in the BE framework to
think about it. From your statements I (your students)
could believe
that if I take my dose of drug A today it could be
effective but WHO knows what will happen tomorrow when
I take again my tablet of drug A...because even if I
reach with the second dose the same plasma levels..the
supercomplex events leading to a particular
trasnpor/uptake/metabolism in cell Z could decide
tomorrow to work the reverse and then I will have no
effect...
I guess/hope you find this statement absurd and I
hope/guess this is not what you meant...
BUT this is what could be understood from your
insistence on the "non-existing relationship" betweem
plasma levels and target cells levels....
Marival
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The following message was posted to: PharmPK
I work primarily with cancer issues. In many cases,
when dealing with cancer, the plasma level plays a
poor predictor of efficacy. We are learning about
cellular transport mechanisms which control both the
influx and eflux of the drug at the target. A recent
discovery with PDX (phase II trials - similar to MTX)
showed that the actions of the drug was dependent only
on cellular mechanisms. We did short trial with
Probenecid to reduce the eflux.
At one point we studied the effect of ascorbic acid
which - if we could stimulate its transport we could
block cell replication. It worked invitro for HIV. But
this transport was dependent on cellular enzyme
capacity to change it to DHAA which easily
transported. Certian Breast Cancer models actually
sequestered the vitamin C which enhanced their
resistance to other chemotherapy.
A better kinetic model is needed for cancer therapy
than just the provision of a plasma level.
Mark G. Klang MS, RPh, BCNSP
Supervisor, Research Pharmacy
Box 538, RL 747
Sloan-Kettering Institute
New York NY 10021
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The following message was posted to: PharmPK
Mark,
By "a better kinetic model" I assume you mean that there is a
relationship, however obscure and indirect it may be, between plasma
concentrations and efficacy. If so, I agree wholeheartedly.
I think that if a tissue receives its supply of drug from plasma, then
there MUST be a cause-effect relationship between plasma concentrations
and pharmacodynamic effect. It may be complex, including
time-dependencies from saturable mechanisms, "hysteresis" effects from
inducing or inhibiting its own transport and/or metabolism, changes in
the tissue as a result of successful therapeutic effect (tumor
shrinkage), etc.
But just because it's complex and we haven't found it yet doesn't mean
it doesn't exist.
Walt Woltosz
Chairman & CEO
Simulations Plus, Inc. (AMEX: SLP)
1220 W. Avenue J
Lancaster, CA 93534-2902
U.S.A.
http://www.simulations-plus.com
Phone: (661) 723-7723
FAX: (661) 723-5524
E-mail: walt.-at-.simulations-plus.com
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