Here - Stiftung Forschung 3R

The final outcome of the work in WP5
was that the corrections for the kinetic
parameters led to an improvement of the
correlation between in vitro data based on
EC 50
s for basal cytotoxicity (3T3/NRU test)
with data on LD 50
s (R 2 values increased
from 0.34 to 0.55 on the inclusion of
kinetic parameters). The algorithms
developed were used in the prevalidation
exercise. A drawback, however, was the
fact that it was not possible to have in vitro
or in silico data for all compounds. This
was mainly due to the lack of sufficient
analytical techniques.
Metabolism
New strategies to incorporate metabolic
capabilities into cell lines
Recombinant-defective adenoviral
vectors encoding for major CYP genes
(CYP 1A2, 2A6, 2E1 and 3A4) involved
in foreign compounds metabolism were
transfected into hepatoma cell lines. In
order to obtain functionally metabolising
cells, other drug metabolising enzymes
(such as cytochrome reductase, heme
synthesis, etc.) must be expressed. Assay
miniaturisation was also accomplished,
allowing the use of 96-well plates and
decrease the virus amounts necessary,
thus making the process more time- and
cost-effective and amenable to HTS toxicity
platforms. The goal was to demonstrate
the applicability of the method developed
as an in vitro screening tool to study CYP
metabolic-dependent toxicity.
Metabolic Stability of ACuteTox reference
chemicals in hepatocytes & microsomes
In vitro half-life and intrinsic clearance
of compounds on the ACuteTox list have
been analysed, using rat liver microsomes
(15 compounds analysed), human liver
microsomes (32 compounds analysed),
primary rat hepatocytes (15 compounds
analysed), and cryopreserved human
hepatocytes (21 compounds analysed).
Data were expressed as in vitro clearance
(in µl/min/mg microsomal protein or µl/
min/10 6 cells) as well as calculated Clint,
hepatic clearance and extraction ratio
using established scaling factors. Species
differences as well as large differences
between the metabolic stability in the
presence of liver microsomes and
hepatocytes were found for some of the
compounds. Data on protein binding in
human plasma for 29 compounds using
microdialysis and LC-MS-MS were also
generated.
Evaluation of computer-based prediction
models for toxicity combining in vitro data
on toxicity and PBBK-TD modelling
The results obtained support the
conclusion that PBBK modelling is a
promising tool that is likely to improve the
possibility to predict toxic doses after oral
administration based on in vitro data, but
also that it is reasonable to question the
approach of using only basal cytotoxicity
data for performing such predictions.
Recalculations were made using the
permeability converter included in the
computational model GastroPlus.
PROGRESS REPORTS FROM EU-FUNDED PROJECTS
Progress Report 2011 & AXLR8-2 Workshop Report
31