WORKPLAN
Workpackage 5
WP5 Screening tests
WP LEADER SUN YAT-SEN UNIVERSITY
Objectives
a) Absorption screening via Caco-2 monolayers in vitro. Caco-2 cell is a widely used method for high throughput screening in intestinal absorption potential of new drug candidates. b) Screening test with animals c) Screening test with viruses
Description of work
The work will involve standard biological techniques by a single postdoctoral co-worker. a) The test will be conducted on available new NA inhibitors prepared by the TAMIFL.OR partner through the domino reactions. Three task will be realized in this workpackage.
Task1: Preclinical screening with Caco–cells. The apparent permeability coefficient (Papp) will be calculated to predict the absorption potential of the test compounds. Test compounds with acceptable permeability will be chose for the next stage of drug development. To establish and validate the analytical method for determination of the target compounds in cell culture mediate; responsible for the cell culture; to conduct the transport experiments via Caco-2 cell monolayer for absorption potential screening. The results obtained with the screening will be used in the implementation of the test conduced with animals
Task 2. Test with rats. In order establish and validate the analytical method for determination of the target compounds in biological samples, and to establish the analytical method for identification of the possible metabolites of the target compounds. Moreover we will focus our attention to carry out the in vivo absorption study, tissue distribution study, metabolites identification study, and excretion study in laboratory animals.
Task 3. Test with viruses. Contemporary to these study, we will try to investigate the effects of different inhibitors on influenza virus replication in MDCK cells will be determined in parallel by an ELISA method that assay expression of the viral NP in infected cells. The second strain in each pair is the reference strain of influenza A virus used for the antigenic subtyping of the NA of the new influenza isolates. The 50% effective concentration (EC50) of the inhibitors will be determined by plotting the percent inhibition of virus replication after correction for background values (obtained from uninfected cultures) as a function of compound concentration calculated from the dose-response curve. Data will be expressed as the means of the EC50s.
Deliverables
A Single report (D10), at month 27, will be provided detailing with the characterization and results obtained in the screening. After other two reports (D11) and (D12) will be provided at month 36. Details of the journal publications realised and planned from workpackages. Aspect of this deliverables will be presented in the public domain via the web interface and the scientific part of the project symposia (D16)