| Jun
1, 2005 |
| By:
Yu
Suen, Konstantin
Tsinman, Zhu
Zhu, Graham
Threadgill |
| Pharmaceutical
Discovery |
|
Early drug discovery ADME assays, such as fast Caco-2 screens, can
help in rejecting test compounds that lack good pharmaceutical profiles. A
cost-effective, high-throughput method — parallel artificial membrane
permeability analysis — that uses a phospholipid artificial membrane
that models passive transport of epithelial cells, is becoming
increasingly popular. The pION PAMPA Evolution 96 System (with Double-Sink
and Gut-Box) is a new surrogate assay that predicts the gastrointestinal
tract absorption of candidate drug molecules at different pH conditions.
This paper describes Beckman Coulter's Biomek FX Single Bridge Laboratory
Automation Workstation PAMPA Assay System that features a 30-minute
incubation time using an on-deck integrated Gut-Box and a SpectraMax
microplate spectrophotometer. The permeability coefficients of drug
standards with diverse physiochemical properties were compared from both
PAMPA and Caco-2 assays automated using the Biomek FX Workstation. These
automated assays can be used for high-throughput ADME screening in early
drug discovery.
Introduction Incorporating
predictive ADME assays in earlier stages of drug discovery can help in
rejecting molecules that lack necessary pharmacological properties. Drug
bioavailability is influenced by factors including absorption and
metabolism. The U.S. FDA issued the Biopharmaceutics Classification
System, which is the guidance for using in vitro models to assess
drug bioavailability. The parallel artificial membrane permeability assay
(PAMPA) using membranes coated with a concentrated mixture of
phospholipids in dodecaine has been widely accepted as a surrogate model
for high-throughput permeability assays measuring passive transport.
Figure 1. The Biomek FX-ADMETox
Workstation with integrated pION Gut-Box and Molecular Devices'
SPECTRAmax 384 Plus.
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The Biomek FX Laboratory Automation Workstation was used to automate the
PAMPA Evolution 96 permeability assay. The PAMPA assay with Gut-Box and
SpectraMax Plate Reader (Molecular Devices, Sunnyvale, CA, USA) is
integrated on the Biomek FX Laboratory Automation Workstation (Figure 1).
Drug permeability and membrane retention for seven reference drugs of
different physiochemical properties were determined at different pH
conditions and are reported here. The rank order of these reference drugs
match well with the conventional Caco-2 permeation assay. The automated
PAMPA Evolution 96 assay can be used for high-throughput primary screening
of drug candidates for ADME properties.
Figure 2. A PAMPA Sandwich Plate
System for the determination of drug permeability through a
phospholipid mixture under different pH conditions.
|
Experimental Conditions All
liquid-handling steps for the PAMPA assay are performed on the Single
Bridge Multichannel Biomek FX Laboratory Automation Workstation, driven
from pION's (London, UK) PAMPA Evolution 96 Command Software. The PAMPA
Evolution 96 Permeability Assay Kit includes System Solution, Acceptor
Sink Buffer (ASB), Double-Sink Lipid Solution and a PAMPA Sandwich plate,
preloaded with magnetic disks (Figure 2). Test compounds used in this
analysis include 10 mM stock solutions of ketoprofen, verapamil,
metaprolol, carbamazepine, ranitidine, propranolol and atenolol. The PAMPA
assay is initiated by transferring 3 µL of lipid to the support membrane
in the acceptor well, followed by addition of 200 µL of ASB (pH 7.4).
Then, 180 µL of diluted test compound (50 µM in system buffer at pH 5.0,
6.2, 7.4 and 8.5) is added to the donor wells.
The PAMPA sandwich plate is assembled and placed on the Gut-Box for the
course of the assay. The Gut-Box generates a rotating magnetic field that
creates an aqueous boundary layer in the donor wells that mimics the
condition in the human gut and shortens the required incubation time for
the assay from 4 h to 30 min. The distribution of the compounds in the
donor and acceptor buffers (100 µL aliquot) is determined by the measure
of the UV spectra from 200 to 500 nm using the SpectraMax reader. The
permeability coefficient is determined using the maximum absorbance from
200 to 500 nm using the following formula:
Pe = 2.3VD/[A
(t-tLAG)]log10{1/(1-R)·CD (t)/CD(0)}
Figure 3. UV spectral scans of
ketoprofen at different pH levels and a plot of the calculated Log
Pe vs. pH.
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Where VD is the donor well
volume (cm3); A is the filter area (cm2); CD
(0) is the sample concentration in the donor well at time 0 (mole/cm3);
CD (t) is the sample concentration in the donor well at time t
(mole/cm3); t is the interval of time (sec); tLAG is
the lag time needed to reach steady state conditions (sec) and R is the
membrane retention (related to the membrane/water partition coefficient).
Table I. A PAMPA assay, that
differentiates drugs with optimal permeability coefficient at
different pH conditions
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Results We
show the ultraviolet spectral scans of a test drug compound from replicate
wells and multiple pH conditions in Figure 3. The scans are overlaid to
demonstrate the reproducibility of the data and to enable the
identification of outliers. As shown in the summary table (Table 1), the
PAMPA assay differentiates the permeability coefficient of the test
compound drug standards under different pH conditions. The rank order of
the drug standards correlates with that determined using a Caco-2 system
on both the Biomek FX and the Biomek 3000 (data not shown).
Conclusions The
Double-Sink PAMPA permeability assay mimics in vivo conditions by
the use of a chemical sink in the acceptor wells and pH gradient in the
donor wells. The use of the pION Gut-Box integrated on the deck has
shortened the PAMPA assay incubation time to 30 min.
The permeability coefficient and rank
order of the seven test drug compounds correlates with data obtained using
the in vitro Caco-2 assay and in vivo permeability
properties measured in rat intestinal perfusions. The automated
Double-Sink PAMPA permeability assay, using the Biomek FX Workstation, can
be used for high-throughput ADME screening in early drug discovery.
Beckman Coulter Inc.
4300 N. Harbor Blvd.
Fullerton, CA 92834-3100 USA
Tel. 800-742-2345; Fax 800-643-4366
www.beckmancoulter.com
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