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Hi,
Please forgive the naivete of this question. Any of you who have
read my posts in the past, know that I have no formal training in PK
and/or chemistry (I'm a biologist); however, this group has been
incredibly helpful to me in working through several problems - Thank
you....
I have recently been given 4 compounds that an investigator wishes to
ultimately test in vivo. These compounds were chosen based on in
vitro activity. I am trying to find a formulation to use now that
can be translated later on into something that can be used in vivo.
I have searched the PharmPK archives and gotten some ideas of
excipients to use, but practically speaking how does one approach
this issue? My naive approach has to been to assume that I would
ultimately want to dose up to perhaps 25 mg/kg in mice (roughly 550
micrograms in a volume of 200 microliters assuming a mouse weight of .
022 kg). This means each compound should be soluble in a workable
formulation up to a final concentration of around 3 mg/ml (rounding
up). I have small aliquots of each compound which I have been trying
with various formulations, but so far nothing has worked very well.
I have tried:
10% DMSO diluted into pH 7.5 saline
5% Tween 80 diluted into pH 7.5 saline
10% 1:1 cremaphor:ETOH diluted into pH 7.5 saline
The LogP values for these compounds range from 0.8 to 4.5 (the 0.8
compound looks to be almost soluble but not completely in the above
mixtures; everything else crashed out fairly convincingly).
Can I vary the pH of my vehicle? What other excipients should I try
and how should these be used? Am I approaching the whole issue
incorrectly?
I'm part of an academic institution with some synthetic organic
chemists around but no one who really thinks about formulation. I'm
going to try talking with at least one of them, but if anybody had
any suggestions, I would be very grateful. Also if there are any
books or references (I'm really looking for protocol type books) that
would shed light on this issue, that would also be helpful.
Thanks,
Noelle
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The following message was posted to: PharmPK
I have personally worked through these same types of formulations
issues. Formulation issues at the research level tend to be the most
overlooked issue when it comes to testing compounds both in vitro and in
vivo. When I was attempting to solve the formulation problems I was
encountering I found the following articles to be of some help:
(1). S.H. Yalkowsky et. al. Formulation-related problems associated with
intravenous drug delivery. J of Pharmaceutical Sciences 87 (7) 787-795,
1998.
(2). Patrick Crowley et. al. Excipients for Pharmaceutical dosage forms.
Encyclopedia of Pharmaceutical Technology, 1151 - 1161, 2002.
(3). Sandeep Nema et. al. Excipients - their role in parenteral dosage
forms. Encyclopedia of Pharmaceutical Technology, 1164 - 1187.
(4). Leon Shargel et. al. Biopharmaceutics. Encyclopedia of
Pharmaceutical Technology, 82 - 102.
(5). P. Montaguti et. al. Acute Intravenous Toxicity of Dimethyl
Sulfoxide, Polyethylene Glycol 400, Dimethylformamide, Absolte ethanol
and Benzyl Alcohol in inbred mouse strains. Arzneim-Forsch/Drug Res 44
(1)566 - 570, 1994.
(6). P.P. Singh et. al. A Pharmacological Study of Propane-1,2-diol.
Arzneim-Forsch/Drug Res 2 (11) 1443 - 1446, 1982.
What I have found is that many investigators fail to ascertain whether
or not their compounds are stable and soluble at the intended tested
concentrations. First of all one must make sure that the dilution schema
used in all in vitro assays are consistent throughout and that a
Nephaloskan or HPLC method is used to make sure that all compounds are
stable and in solution. Because one typically uses a higher
concentration of compound for in vivo studies, it is really imperative
that some analytical testing be performed prior to testing the drug, to
ensure the drug is still in solution (this cannot always be assessed by
visual inspection of the material).
What happened in my case was once I noticed the drugs I was given to
test in vivo were precipitating out of solution, we went back and tested
the samples used in the in vitro assays, and alas, these compounds were
also precipitated out of solution. Believe me, solid material coated on
cells kills cells very nicely, even in a titratable manner.
I ended up doing a large in vivo tolerability assessment of various
excipients (DMSO, PEG-400, cyclodextrins, alcohol, cremophor,
tween-20/80) alone using various volumes, various injection schedules,
in different strains of mice, using two administration routes (iv and
ip). My drugs were only soluble and stable in 80 - 100% DMSO. I found
that all my strains of mice tolerated up to 50 uLs 100% DMSO
intraperitoneally q1d x 5.
What would have been ideal would have been to do an oral bioavailability
study of these drugs, but that was not in the plans for this project.
Hope this helps.
Jennifer
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Noelle:
The choice of vehicle and pH is depend upon the route of administration
in the experimental animals. If you know the Pka of chemical, there is
a good chance that by simple manipulation of pH adjustment (insitu salt
formn) you may be able to achieve adequate aqueous solubility.
However, these solutions may precipitate upon dilution in vivo due to
difference in GI pH (based on whether drug is acid or base) and you
have no control over the kind of precipitate it forms invivo. If you
want to evaluate oral route, it is advisable to use simple micronised
drug in CMC with small amount of surfactant (0.1% to 0.25%) such as
tween 80. If you already know that oral bioavailability is poor and it
is dissolution/solubility and not permeability/metabolism it is worth
spending time on slightly complex formulations such as complexation
with cyclodextrins, lipids or cosolvents with or without pH adjustment;
pH of 2.0 for IV use in experimental animals (multiple dose) is not
uncommon.
best regards
shanthakumar
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Hi
We have tried PEG 200/saline/ethanol in the ratio of 40:50:10 and its
works resonabbly well for most compounds in a discovery setting. Of
course, you cannot use this formulation for a dog PK. An other
approach that we have used is citrate buffer at a pH of 4 (if you have
some basic moieties in your compounds).
I hope this helps.
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The following message was posted to: PharmPK
Dear Noelle,
playing with the pH of the formulation is usually a powerful tool to
solubilize ionizable Compounds.
The first question would be: do you know the molecular structure of the
compounds, so you would know if and how they could be ionized.
Second question: how much compound do you have in hand to test various
formulations. For skilled (and equipped, e.g. microskope) formulation
scientists, 10mg is a wealth. Often, they have to deal with much less
than
that.
Third and last: how do you want to apply the compounds? For oral
application, you don't necessarily need a solution, a homogenous
suspension
could be a good alternative.
For an overview, look at Pfizers decision tree published by Lee et al.:
International Journal of Pharmaceutics 253 (2003) 111- 119, if you
want to
search the lit by yourself, Samuel Yalkowsky published many papers on
this
topic.
good luck,
Philip
*************************************
Philip Lienau
Schering AG
Research Pharmacokinetics
Tel.: +49 - 30 - 468 - 18507
Fax: +49 - 30 - 468 - 12238
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The following message was posted to: PharmPK
Jennifer,
please keep in mind that DMSO is an excipients that strongly modifys the
barrier functions of tissues. Performing bioavailability studies
using this
solvent (orally) could thus lead to false positive results (and often it
does).
So, looking for the right formulation is not only a problem of
solubilizing
a compound but also to generate an inert formulation.
If you want to check the Lit. on this topic, Beate Bittner from Roche
published very good articles (including excellent reviews)
best wishes,
Philip
*************************************
Philip Lienau
Schering AG
Research Pharmacokinetics
Tel.: +49 - 30 - 468 - 18507
Fax: +49 - 30 - 468 - 12238
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The following message was posted to: PharmPK
Hi Noelle,
preclinical formulation issues are always relevant questions to
ask !!! Don't be afraid to be seen as naive. There is no reason for
that.
Regarding your information request, I would like to add a few
comments and ask some questions :
As far as I understand your message you would like to perform some
"proof of concept" studies. By this I mean that your objective is to
get the max amount of drug in the mice. In this case your approach is
not that bad. You should try and find "exotic" formulations to ensure
that your drug is in the mice. But please keep in mind that this kind
of formulation should not be used for further preclinical studies
(PK, ED50, ...).
Regarding logP data I am convinced that those drugs should get some
water solubility (obviously not up to 3 mg/mL !!!). Do you have any
value ?
What is the route of administration you intend to use ? IV ? IP ?
PO ? SC ? other ? The approach is quite different.
The trouble with all you tried (co-solvent approach) is the risk of
precipitation (as described by people who replied to your message).
Jennifer Thompson used 100% DMSO but I am concerned that this led to
some massive precipitation in a water environment.
Please give more information (water or pH 7.4 buffer solubility and
route of administration) and I try and design a relevant approach.
Best regards,
Frederic Doc
ACRITER - Drug discovery consulting
www.acriter-consulting.com
Former preclinical formulation scientist at PFIZER
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The following message was posted to: PharmPK
Phillip,
Apparently I did not make myself clear in my last email. I wasn't saying
that I wanted to use the DMSO formulation for oral administration. What
I would have preferred to do, in the case of a highly insoluble drug,
was to prepare in a less harsh/more inert formulation for oral
administration. Because of the time line of the project I determined
that formulating my drugs in 100% DMSO and administering them ip was the
only option.
Thank you,
Jennifer
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Dear Noelle,
The best and simple approach to start with the solubility
studies of NCEs would be checking its aqueous solubility in water and
either with Normal saline or pH 7.4 buffer. If it's not soluble, then
look at the structure for any ionizable group (Many a times in-silico
tools would be handy in prediction of pKa and such relative
parameters). If it is an ionizable compound, then you may try with pH
manipulation using buffers viz., acidic groups can be ionized with
basic buffers etc. to acieve the solubility. If either there's no
ionizable group in your NCE or there's a precipitation upon dilution
and you intend to administer it orally, then you can make up a simple
suspension with 0.15-0.25% Tween 80 ( Be cautious in using Tween-80 as
it is a substrate of P-Glycoprotein, an efflux transporter) and 0.25%
CMC. Most of the NCEs would work in making uniform suspension with thi
s approach.
If your objective is still to administer it in solution form
either in oral or i.v, then use lipid or organic co-solvents (Opined by
many of PHARMPK members) to acheive a solution form. If nothing works
out, then you can attempt the complexation with cyclodextrins or
modified CDs like Captisol (Voriconazole and Ziprasidone have already
been marketed with Captisol formulation by Pfizer) or with Vitamin E
TPGS USP or with Pharmasolve etc., However While using the complexed
formulation, be cautious in interpretation of PK parameters of your
NCEs
since in-vivo release pattern may be so different that can alter the PK
parameters.
Hope this helps.
Regards,
Syed Mustafa,
Dept of Pharmacokinetics and Metabolism,
Orchid RND centre,
Plot No: 476/14,
Old Mahabalipuram Road,
Sholinganallur,
Chennai : 600119
syedmustafa.aaa.orchidpharma.com
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Dear Noelle & Group,
There was a typographic error in my response. Please read it as
follows: Tween 80 ( Be cautious in using tween-80 as it is an inhibitor
of P-Glycoprotein, an efflux transporter), which by mistake appeared as
a substrate of P-gp. PHARMPK group to excuse for the same.
Regards,
Syed Mustafa
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The following message was posted to: PharmPK
Hi all,
My query is to Jennifer Thomson where she used 100% DMSO for
invivo studies. More or less i have a similar situation as her, my
drug is
precipitating out during my invitro studies.
what could happen invivo if i give 100% DMSO-Drug IP dose to a mice?
will the
drug precipiate during invivo studies too? will there be any
immediate side
effects because of 100% DMSO and also due to the precipitated drug??
i am not a pharma guy, and these insoluble drugs taking a lot of time
to produce
results.
thank you for your help.
Rajesh Kumar
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The following message was posted to: PharmPK
Dear Rajesh,
I am concerned that precipitation should occur after ip dosing of a
100% DMSO formulation.
And obviously you should also see tox effects of this vehicle in mice.
Of course insoluble drugs are a key issue in drug discovery.
A relevant preclinical formulation strategy should be designed in
order to enhance the productivity of research activities. My group
developed such a strategy when I was at Pfizer. I now offer such
services as an independent conslutant.
Best regards,
Frederic Doc
Acriter - drug discovery consulting
www.acriter-consulting.com
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The following message was posted to: PharmPK
You might want to use other less irritant solubilizing agents
such as cyclydextrins or polyhydrogenated castor oils (the
Cremophor Family). The latter is widely used in vivo IP and
IV.
Murad
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The following message was posted to: PharmPK
Rajesh,
These are very good questions you ask. I asked the same questions at the
time I performing these assays. Prior to doing any in vivo studies, I
tested a large subset of my compounds with various excipients (DMSO,
PEG, cyclodextrins, etoh, cremophor) alone and in different combinations
with one another. The only excipient that solubilized the majority of my
compounds was 100% DMSO. I then designed and tested a large in vivo
excipient tolerability study with numerous excipients alone and in
combination with one another. I found no effect (no weight loss,
etc...), on numerous mouse strains, of 50 uLs DMSO injected ip q1d x 5.
Of course, there's always the possibility that the drug will precipitate
out of solution once injected. As a matter of fact I'm sure that the
drugs I injected precipitated out of solution upon being injected. My
initial dilution studies showed that the instant I diluted my drugs from
100% DMSO to 95% DMSO, whether or not I was diluting in 100% mouse
plasma or serum or 100% PBS, the drugs precipitated out of solution. I
presented this information to the people in charge of this project, but
they had no formulation experience and did not realize the limitations
this placed on the success of the project. The people in charge decided
to continue formulating the drugs in 100% DMSO and injecting them ip q1d
x 5.In my case, even though I knew the drugs were more than likely
precipitating after injection, sometimes I saw toxicity (>15% weight
loss, acute death, etc...) and sometimes I saw no toxicities.
Companies/Institutions that are smart and have integrity will listen to
concerns about formulation issues because they realize how important
having a properly formulated compound is for a successful drug discovery
projects. Hopefully the company/institution you work for will allow you
to investigate what formulation is appropriate for your drugs.
In regards to your in vitro assays, the dilution schema you use for your
in vitro assays is incredibly important to whether or not your drugs
will precipitate out of solution. In my case, the first dilution step,
going from 100% vehicle to the next percentage was the most critical
part of diluting drugs for in vitro assays. We used a Nepheloskan and
HPLC to determine whether or not the compounds were precipitating out of
solution. Once I determined which small dilution I needed to do
initially, I could, from this dilution, do a large dilution and my
compounds remained in solution.
Hope this helps.
Jennifer
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I would like to pose a question- if a compound is so water insoluble
that most of it precipitated out in ip injection, is it worth it to
develop the drug? Or is it better to go back to screen for more water
soluble drug.
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I agree with this respondent. If the compound is soluble only in DMSO
and is almost like sand/ rock, does it have any future. Moreover , do
you have this problem in the whole series or just the compound in
question.
Sunita
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Jennifer,
I read your message with a lot of interest.
It is sometimes very hard to convince in vivo scientists that the
relevance of preclinical drug dosage forms is key to enhance the
confidence in experimental data.
You are right when saying that "smart" companies/institutions will
listen and focus on solving formulation issues at early stages of
drug discovery. This is the reason why I propose consulting services
in "preclinical drug formulation strategy design and implementation".
We have some contacts and clients (some of them are part of the
PharmPK list) we are collaborating with on this topic.
Best regards,
Frederic Doc
Acriter - Drug discovery consulting
www.acriter-consulting.com
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The following message was posted to: PharmPK
Very very important question !
As a preclinical formulation scientist my first answer would be :
"no! If your drug is such a rock, just drop it and get a new one."
But as a research project leader I would answer that this should be
balanced with other data. This is the reason why research projects
often use parameters and limit values (does everyone understand if I
use the term "cut-off"?) to select compounds in "lead optimisation"
or "candidate seeking" phases.
Well I am convinced that very insoluble chemicals cannot be handled
to become relevant drugs as they exhibit variability in PK data and
biological effects as their solubilization (prior to any absorption
or distribution) is highly dependent on in vivo conditions: for
example if we consider a PO dosing I would not be surprised that one
observes very different data whether the drug is dosed in a fed (low
fluid volume, low amount of bile salts) or fasted state (higher fluid
volumes and a higher amount of
solubilizers).
Does it make sense,
Best regards,
Frederic Doc
Acriter - drug discovery consulting
www.acriter-consulting.com
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Hi All,
Thx for your comments. Jennifer I am trying my level best make my
Supervisor listen to the issue. Right now spinning my head for an
invivo expt, ofcourse will ask for Frederic's help when needed.
Regards
Rajesh
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The following message was posted to: PharmPK
Very very important question !
As a preclinical formulation scientist my first answer would be :
"no! If your drug is such a rock just drop it and get a new one."
But as a research project leader I would answer that this should be
balanced with other data. This is the reason why research projects
often use parameters and limit values (does everyone understand if I
use the term "cut-off"?) to select compounds in "lead optimisation"
or "candidate seeking" phases.
Best regards,
Frederic
Frederic Doc
ACRITER - Drug discovery consulting
www.acriter-consulting.com
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Dear Frederic,
Before dropping a rock-like drug, I would consider to perform
solubility
test(s) with methylated Beta-cyclodextrins. Based on my experience
on solubilization, the 2,6-di-O-methyl derivative (DIMEB) always
presented the best solubility enhancement, and it actually happenned
that it was the only working cyclodextrin. Freely water soluble
easy to
hand pre-formulations with more or less active content can easily be
obtained for in-vivo testing.
I think, in case of such drug candidates, low dose is a very
important cut-off parameter for further selection.
Best regards,
Maria Vikmon, former researcher at Cyclolab, Cyclodextrin R&D Ltd,
Budapest, Hungary
vikmon.aaa.freemail.hu
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The following message was posted to: PharmPK
Dear Maria,
coming back to my previous message, I would comment that dropping a
rock-like drug may be the point of view of a formulation scientist.
A research project leader would balance the low solubility with the
efficient dose.
On the point of cyclodextrin use I did a lot of work with beta-
hydroxypropyl-cyclodetrin. We used this ingredient in setting up iv
formulations. Being certainly some of the best solubilizing agents
cyclodextrins have to be used carefully as they complex with poorly
water-soluble drugs. This leads to get a focus on release kinetics to
ensure that in the plasma the drug can freely go out of the
cyclodetrin cavity to be efficiently distributed.
Last point : rock-like drug and cyclodextrin. A relevant preclinical
formulation strategy is needed in each organisation to ensure that PK
data are usable and reproducible. And cyclodextrins are part of this
formulation strategy (at least they were part of the strategy we
implemented at Pfizer!).
Best regards,
Frederic Doc
ACRITER - Drug discovery consulting
www.acriter-consulting.com
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