omation mbers - Society for Laboratory Automation and Screening

WP064
Rowan Stringer
Novartis Horsham Research Centre
Biology 1
Wimblehurst Road, Horsham, West Sussex
Nr LondonRH12 5AB United Kingdom
rowan.stringer@pharma.novartis.com
96-Well Caco-2 transport Model for Drug Permeability Studies
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Co-Author(s)
Elizabeth Willmott
Rachael Profit
Sarah Beech
Paul Nicklin
A 96-well Caco-2 permeability screen has been established for drug absorption profiling. After 21 days of culture
on Millipore MultiScreen ® inserts, light microscopy showed the formation of intact monolayers of polarised
columnar cells. Ultra-structural studies confirmed the presence of a microvilli brush-border and tight junctions
between cells. Monolayers had low permeability towards the paracellular marker, [ 14 C]mannitol (Papp = 0.21±0.06
cm/s), and high permeability towards propranolol (Papp=19.6±4.6 cm/s). This system had good intra- and interplate
reproducibility for reference compounds (e.g., CV = 18% for the Papp of 15 drugs, over 16 different test
occasions). Moreover, a good correlation was observed between their in vitro Papp and the fraction absorbed
in man. The Caco-2 monolayers were also Pgp-competent, having a net efflux transport for known substrates –
e.g., efflux ratios for acebutolol (~100-fold), quinidine (~20-fold) and verapamil (~3-fold). The 96-well screen
was integrated with an automated bioanalytical platform, enabling high compound throughput. For example,
a set of 100 marketed drugs and 1200 discovery project compounds were screened in 16 weeks. This model
system differentiated compound classes having low and high permeability as well as highlighting those having a
susceptibility for efflux transporters.
WP065
Michael Su
Molecular Devices Corporation
Instrument R&D
1311 Orleans Drive
Sunnyvale, California 94089
Michael_su@moldev.com
Co-Author(s)
Jinfang Liao
Evelyn McGown
Dual-Glo Luciferase Assay Measurements in the LMax II 384 Microplate Luminometer
and the Analyst ® GT Multimode Reader
Reporter gene assays are used to study eukaryotic gene expression. Dual genetic reporters are commonly used in
transient transfections of cultured cells to minimize experimental variability caused by differences in cell number,
viability or transfection efficiency. One plasmid containing the experimental reporter gene (coupled to a regulated
promoter) is cotransfected with a second plasmid containing a control reporter gene (coupled to a constitutive
promoter). Bioluminescent reporter systems using firefly and Renilla luciferases are widely used as co-reporters
because both assays are easy and sensitive. Previously, luciferase reporter assays have been “flash” assays that
must be read within seconds of reagent addition and require integrated injectors in the luminometer. Recently,
Promega introduced a Dual-Glo Luciferase assay System for high throughput analyses. The signals are stable for
2 hours after reagent addition and integrated injectors are not necessary.In this study, we optimized measurement
parameters and compared Dual-Glo Luciferase assay results on the LMax II 384 Microplate Luminometer and
the Analyst ® GT Multimode Reader. Due to the stability of the luminescent signal, both instruments are well suited
for integrating this dual luciferase assay into high throughput screening lines.
POSTER ABSTRACTS