Back to the Top
Dear All,
For a screening program, we want to use the procedure of cassette
dosing in mice. As the objective of this procedure is to characterize
the PK profile of various molecules at the same time, I am concerned
about the validity of the PK data (Interactions at different levels of
ADME). Does anyone have experience with this procedure? What are the
main points to consider?
Your help is greatly appreciated,
Thanks,
--
Samia Ezzine, D.Pharm, M.Sc, Ph.D
Pharmacokinetic Scientist
Preclinical Research and DMPK
Neurochem Inc.
275 Armand Frappier Boulevard
Laval, Quebec, Canada
H7V 4A7
Tel: (450) 680-4441
Fax: (450) 680-4505
sezzine.-at-.neurochem.com
Back to the Top
Samia Ezzine,
High through put in-vivo screen known as 'cassette dosing' or 'n in
one dosing' has since long been used to rank molecules. The attractive
features of the screen include highthrough put; reductions in animal
usage, lesser inter animal variation and a single consolidated report.
The fact that the technique has not gained enough popularity over the
years is a clear indicator of some inherent practical difficulties of
the technique. While administering a large number of compounds together
in the same animal there is a large possibility of DDI . There could be
false positives due to Cytochrome P450 (CYP 450) inhibition. False
negatives due to P glycoprotein (Pgp) inhibition in brain are also
common, which implies that in a cassette due to the inhibition of Pgp
by a co-administered compound, the test compound appears in brain,
which is not the case when administered as a single compound. Compounds
with high protein binding could encounter competitive protein
displacement, which would project a deceptive picture of high plasma
concentration. Moreover, the time required for developing a
bioanalytical method capable of accurately estimating several compounds
in a single chromatographic run is enormous. The third challenge is the
preparation of a single usable formulation containing several molecules
which is often an arduous task to accomplish.
To add reliability to a cassette data , the number of compounds per
cassette can be reduced to 5 and the dose size can be reduced to the
lowest quantifiable dose. It is always advantageous to use an internal
standard to the cassette for verifying the PK results.
An alternative to cassette dosing is a technique known as 'cassette
analysis' . This technique requires pooling of plasma samples from
individually dosed animals dosed with a single compound . Cassette
analysis has the advantage of producing more reliable pharmacokinetic
data as compared to cassette dosing since it is devoid of drug -drug
interactions.
Another popular technique is 'rapid rat' screen where rats (n=2 )
are dosed with a single compound and plasma is collected at equal time
intervals of I h over a period of 6 h. The plasma samples collected
from each of the time points of an individual rat are then pooled and
analyzed using a miniature standard curve.
Please refer to the following references:
1.White R.E and Manitpisitkul P (2001) Pharmacokinetic theory of
cassette dosing in drug discovery screening. DDT . 29(7) , 957-966
2.Christ D. D (2001) Cassette dosing Pharmacokinetics: Valuable tool or
flawed science? Drug Metab. Dispos. 29(7), 935.
3.Manitpisitkul P and White R.E (2004) Whatever happened to cassette
dosing Pharmacokinetics ? DDT. 9(15), 652-658
4.Cox K. A et. al (1999) Novel in-vivo procedure for rapid
pharmacokinetic screening of discovery compounds in rats. DDT .
4 (5) , 232-237
with best wishes
Dr Sonu S. Singh
Principal Scientist
Drug metabolism and Pharmacokinetics Deptt
Zydus Research Centre
Ahmedabad
Back to the Top
The following message was posted to: PharmPK
Dear Samia,
we have used cassette dosing in rats during a lot of optimisation
programs. Our paradigm is planned to check, before cassette dosing in
rats, inhibition of human CYP1A2, CYP3A4, CYP2D6, CYP2C9, CYP2C19 and
CYP2E1 and CaCo-2 cells permeability. This approach helps us to reduce
possible heavy cross inhibition of CYPs (even if we check human CYPs
and perform cassette dosing in rats)and unwanted low permeability with
or without PGP retro-fluxes contribution.
In my experience one of the major concerns performing cassette dosing
is the inhibition of carrier mediated (PGP, OATP, etc.) clearance (and
disposition) process. In my experience it's easier to find false
positive (i.e. compounds with higher bioavailability after cassette
dosing then after single dosing) and not false negative compounds (i.e.
compounds with lower bioavailability after cassette dosing then after
single dosing).
In any case, false negative compounds are also described.
My personal suggested approach is to start with cassette
pharmacokinetics and then to check PK properties of more interesting
compounds in a single dosing protocol.
Exellent papers critically review cassette dosing theory and scenario:
Ronald E. White and Prasarn Manitpisitkul
Pharmacokinetic Theory of Cassette Dosing in Drug Discovery Screening
Drug Metab Dispos 2001 29: 957-966
David D. Christ
Cassette Dosing Pharmacokinetics: Valuable Tool or Flawed Science?
Drug Metab Dispos 2001 29: 935
Manitpisitkul P, White RE.
Whatever happened to cassette-dosing pharmacokinetics?
Drug Discov Today. 2004 Aug 1;9(15):652-8.
Best regards
Stefano
Dr. Stefano Porzio
Pharmacokinetic and Tox. Dept.
Inpharzam Ricerche SA - ZAMBON-GROUP
Taverne - Switzerland
Back to the Top
The following message was posted to: PharmPK
Dear Semia,
what you should consider is to keep the dose as low as possible. Our
approach is to divide the dose we would administer for a single
compound by
the number of compounds in the cassette. To prove the validity of your
study it helps to put a compound with known PK into the cassette as
"biological standard". What you need in addition is sensitive and
selective
bioanalytics (LC-MS/MS). The compounds showing positive PK results in
the
cassette approach should
be tested afterwards in a mono-study to confirm the results. It is our
experience that interactions in cassette studies are very rare when you
keep the dose low.
I hope this helps.
Best regards,
Bernhard J. Ladstetter, Ph.D.
Vice President, Head of Global Non-Clinical DMPK
Global Preclinical R&D
Institute of Drug Metabolism and Pharmacokinetics
Merck KGaA
Am Feld 32
D-85567 Grafing
Germany
Phone: +49 (0)8092-700810
Mobile: +49 (0)172 8661247
Fax: +49 (0)8092-700899
e-mail: bernhard.ladstetter.-a-.merck.de
mobile +33 622 040 516
eliane.aaa.emf-consulting.com
Back to the Top
Dear Dr. Bernhard and Dr. Sonu,
Thank you for your input in cassett dosing. But I couldn't digest the
logic behind using an internal standard with known PK characteristics.
Is it necessary that your so called biological standard ( With known
PK) will always give valid insight into PK data of compounds used for
cassett dosing? I mean how would you conclude the situation in which
say, there is no interaction between your internal standard and rest
4-5 compunds in a cassett, but compounds in a cassett are interacting
with each other? In this situation, you may not see change in PK of
internal standard, but you may get some false positives and false
negatives for compounds in cassett.
Back to the Top
Dear Dr. Sonu,
Can you put Rapid Rat screen somewhat more elaborative?
Back to the Top
Hello,
Cassette dosing has always rasied the issue of drug interactions. If
it comes to simultaneous dosing of NCE's to the model, this issue
should not be ignored. I have seen a paper which was published during
1999 where in they described a method of rapid in vivo screening of
NCEs and to rank order the coumponds. I am sending that article as an
attachment. Pl. go through it. [Not attached - db]
Regards,
Ravi
[Reference: Cox, K.A. et al., 1999 DDT, 4(5) p232-7 - db]
Back to the Top
The following message was posted to: PharmPK
Dear DMPK research colleagues,
Our group has occasionally used cassette dosing, mainly for ranking
purposes, but for many of the reasons already cited by others, we do not
use it routinely. Formulation issues have been one issue (e.g., the
differential solubility of poorly soluble compounds, precipitation of
one entity when another is added). Another issue relates to the
analysis needed: (e.g., compounds that are structurally closely related
may produce the same major fragment needed for quantification in the
mass spectrometer, more time needed to work out an analytical method).
Whenever more definitive data is needed, we do not rely on cassette
dosing results, but repeat a compound with mono dosing.
We have used cassette analysis, perhaps more often than cassette dosing.
Thomas L. Tarnowski, Ph. D.
Project Team Leader
Dept. Head (Dep), Drug Metabolism and Pharmacokinetics
Roche Palo Alto
3431 Hillview Avenue, Palo Alto, CA 94304
* email tom.tarnowski.aaa.roche.com
Back to the Top
Rapid rat screen
In a rapid rat screen , a single compound is administered to two rats
and blood is withdrawn at equal time intervals of I h over a period of
6 h. Equal volumes of plasma collected at each time points from an
individual rat is then pooled and analyzed using a miniature standard
curve. The pooled plasma concentration is multiplied by a factor of 6/7
to account for volume difference resulting from pooling of plasma from
six time point. The corrected concentration is further multiplied by 6
to obtain the estimated AUC (area under the plasma concentration-time
curve). Individual plasma samples collected at 6 h can be analyzed in
addition to the pooled sample. This provides valuable information
about the half life of the compound i.e., a low plasma concentration at
6 h indicates a short half time and vise versa, without any significant
increase in total analysis time. The rapid rat PK screen is devoid of
DDI's , method development is comparatively easy for a single
compound and analysis time is very short as there are only four
samples per compound. The limitation being that the AUC and half life
obtained are not the actual values but are estimated ones and
absolutely no information about the Tmax (time point of maximum plasma
concentration) and Cmax (maximum plasma concentration) is obtained.
This in-vivo highthroughput technique allows initial selection of
candidates with the desired pharmacokinetic properties, The selected
compounds may further be dosed individually to obtained detailed PK
profile.
For details please refere the reference:
1. Kathleen A Cox et. al (1999) Novel in-vivo procedure for rapid
pharmacokinetic screening of discovery compounds in rats. Drug
Discovery Today, 4 (5), 232-237
please view the soft copy of the reference [Not attached - db]
best regards
sonu
Dr Sonu S Singh
DMPK deptt
Zydus research centre
Ahmedabad -India
Back to the Top
You have rightly pointed out that addition of 'biological internal
standard' to a cassette may not be a fool proof strategy when :
1. The test compounds do not interfere with the PK of internal
standard or
2. The internal standard is metabolized by more than one
drug-metabolizing enzyme and will have a decreased likelihood of
clinically significant drug interactions due to the availability of
compensatory metabolic pathways if one is inhibited.
Therefore prior information on in-vitro enzyme inhibition (CYP 450 &
Pgp ) profile of the test compounds should be obtained . Based on the
inhibition data, a known substrate of a CYP450 or a drug transporter
protein like P-glycoprotein (Pgp) can be chosen . For instance ,
addition of a known substrate of P-gp as internal standard can be
advantageous while conducting brain penetration studies to ensure that
the P-gp have not been inhibited . Similarly , compounds exhibiting
in-vitro CYP3A4 inhibiton can be added to the same cassette and a
substrate of CYP3A4 can be used as internal standard.
It is interesting to note that one hand drug interaction in cassette
dosing in animal models is perceived as a major draw back , but
similar studies in man known as 'cocktail dosing" are in fact
conducted to evaluate the drug interaction potential of a compound
..
Regards
Dr SONU S SINGH
DMPK deptt
Zydus Research Centre
Ahmedabad
Back to the Top
Hi all
Here are few more references where cassete dosing or 'N-in-one" dosing
has been successfully employed to rank the compounds.
LC fluorescence method for multiple synthetic compounds to rapidly
create in vivo pharmacokinetic database utilizing 'N-in-One' dosing.
Rajanikanth M, Gupta RC. J Pharm Biomed Anal. 2001 Nov;26(4):519-30.
Simultaneous quantitative analysis of three drugs by high-performance
liquid chromatography/electrospray ionization mass spectrometry and its
application to cassette in vitro metabolic stability studies.
Rajanikanth M, Madhusudanan KP, Gupta RC. Rapid Commun Mass Spectrom.
2003;17(18):2063-70.
Like Dr.Tarnowski, our group at CDRI, India had issues with
formulation, size of cassette and related issues. I understand that
the concept of Sample Pooling is being successfully applied. May be
someone from Pharmacokinetics & Metabolism Division, CDRI can comment
on it.
Raj
Rajanikanth M
Post Doc Assoc
Pharmaceutics Division
University of Florida
Gainesville
PharmPK Discussion List Archive Index page
Copyright 1995-2010 David W. A. Bourne (david@boomer.org)