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The following message was posted to: PharmPK
One of the hardest issue of "in vitro" experiment is always tring to
dissolve a water-insoluble compound. When I hear of such problems, I
always remind a 1991 elsevier book that probably many of you have in
your collection "Isolated hepatocytes preparation, properties and
application" by Berry MN et al. In this book the authors wrote (page159)
that water-insoluble compounds can be dissolved in acetone (highly
volatile), added to bottom of the empty incubation vial and dried under
N2. Once the hepatocytes are added to the vial, they will easily pick up
the compound from the bottom of the vial.
My question is: do you ever tried and controlled this method?
Based in your experience is always necessary to have medium in which the
compound to test is completely dissolved or is just enough having an
opalescent medium to get results? After all, the membranes are the only
lipidic-phase existing in the medium and the lipophilic drugs will
always have a tendency to dissolve in that phase.
Dr Roberto Conti
Sigma-Tau
Dep. of Endocrinology and Metabolism
00040 pomezia, Roma
Phone: +39-06-91393322
Fax: +39-06-91393988
e-mail. roberto.conti.at.sigma-tau.it
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The following message was posted to: PharmPK
Dear Dr Conti,
That's a good question indeed! When I used to work with primary rat
hepatocytes I always included some bovine serum albumin (BSA) in the
medium and I am wondering whether this is perhaps more responsible for
re-dissolving the compound than the cells themselves. IMO the compound
needs to get into solution (either as free drug or protein-bound) before
it can be taken up by cells. I don't think it is realistic to expect the
cells to actually re-dissolve directly from the solid at "the bottom of
the vial". I guess it will depend on many factors (e.g., stirring, vial
geometry, drug and solid state, etc.) on how rapidly a drug will
re-dissolve and knowing the true drug concentration (as a function of
time) & exposure is of paramount importance.
Currently, we use alphaMEM plus 5% foetal calf serum (FCS) for our work
with anticancer drugs in established (human) caner cell lines. We
routinely test stability & solubility of our compounds in this medium
and have found that the inclusion of 5% FCS can make a dramatic
difference in the solubility of compounds, even without cells.
HTH
Frederik
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 Dr. Roberto Conti,
Further to Frederik's comments, I would like to add that in addition to
"keeping the drug in solution", it also important to know the free drug
concentration in solution. Further, it is important to note that use of
protein (such as BSA) help keep the drug in solution (in bound form).
However, it is the free drug that is "thermodynamically active" and
available for transport across the membrane or interact with a receptor.
Since there will be a dynamic equilibrium between free and bound forms,
the drug that is bound to protein acts as a reservoir. Depending on the
association constant of the drug to given protein, drug and protein
concentrations, there will be a constant free drug fraction maintained.
So, for calculation of any kinetic parameter (such as permeability
coefficient) it is important to quantify the free concentration. Kinetic
parameters obtained based on total drug in solution (free+bound forms)
would be erroneous and under-estimated depending on the affinity of the
drug to a given protein.
I have done some work addressing this issue but using non-ionic
surfactants and developed a simplified approach to address changes in
free drug concentrations (in case of surfactants - it is the
solubilisation of drug into micelles which makes it un-available for
transport across the membrane) in an attempt to estimate true
permeability of poorly water soluble compounds under solubilising
conditions.
I am presenting a poster (No. W4093 in PDD section) on this in the
forthcoming AAPS-2005 annual meeting and exposition to be held in
Nashville during Nov 6-10. I welcome interested members attending this
conference to visit my poster.
I appreciate more insight on this issue.
Thanks,
Kasiram Katneni.
Dept of Pharmaceutics
Vic Coll of Pharmacy
Monash University, Australia.
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The following message was posted to: PharmPK
Dear all,
I agree with Kasiram regarding the exposed key points :
1- I would recommend to completely solubilize compounds for in vitro
experiments moreover when you consider a rapid test (someone in a
very recent PharmPK message talked about performing an 8-minute run).
2- I would also recommend to take into account the bound fraction of
the active (bound to BSA for instance) and the kinetics to "release"
some free drug from the "protein reservoir".
3- In the case of an in vitro experiment the key point is to ensure
that solubilizers do not modify cells. This means that there are at
least two points to check : cell viability and cell membrane integrity.
I did some trials checking cells' behaviour in media containing some
polysorbate. For rapid runs you should be able to find out the right
concentration of polysorbate to be used to solubilize the active
without modifying cells integrity and viability.
I would definitely not recommend cyclodextrins because it would add a
complexation/decomplexation kinetics phase to the previously
described BSA bound/free kinetics one.
Let me end up with a non-expert of in vitro experiment questions :
would it be a good idea to use natural intestinal surfactants such as
sodium taurocholate ? I would expect that they have good solubilizing
properties and they are probably not as agressive for cells as
commonly used solubilizers. Does it make sense ?
Regards,
Frederic
Please visit us at : www.acriter-consulting.com
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The following message was posted to: PharmPK
Hi Frederic,
It is hard to imagine a surfactant (natural or synthetic) to be both
a good solubiliser and not have effect on the cells/monolayer at the
same time. It all depends on the property of the surfactant and
concentration used. For instance, take polysorbate-80 (PS-80, a
synthetic surfactant which got CMC of around 0.05mM) and Sodium
taurocholate (NaTC) a natural surfactant whose CMC is around 5.0mM).
As we know, for a surfactant to solubilise, the concentration should
be above its CMC. Which means that in case of PS-80 the concentration
would be much lower (almost 100 times) than for NaTC. So, it is not
appropriate to compare the effect of a surfactant on cell viability
or monolayer integrity, at equi-molar concentrations (say 0.5mM),
since for PS-80 it is above its CMC and for NaTC it is below its CMC
and as a result PS-80 may show toxic effect and NaTC may not.
The bottom line is that we can not generalise and the applicability
or safety of a surfactant for a specific application had to be
validated prior to its use.
Kasiram.
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The following message was posted to: PharmPK
thanks every body for great help
but still the problem lingers around
a surfactant/ solubilizer will definately modify cells
i found a dose dependant effect with DMSO, and CMC
HPMC however didnot show any effect, so also HPBCD
but then these additives add one more parameter of complexation as
rightly pointed out by Dr.Frederic
i have not tried with polysorbates but still have doubts as the
platelets are very sensitive to slightest of environment
modifications...even if platelet poor plasma is used as solubilizer
still it gives a problem in evaluation
will need more inputs on this topic
kind regards
amrutesh
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