Drug Discovery and Development Technology Center Drug Discovery Early-ADME &preformulation Pharmaceutical Nanotechnology Independent Research Centre in.

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<ul><li>Slide 1</li></ul> <p>Drug Discovery and Development Technology Center Drug Discovery Early-ADME &amp;preformulation Pharmaceutical Nanotechnology Independent Research Centre in the Faculty of Pharmacy Multidisciplinary Networking: Faculty, other Funding Slide 2 DDTC in Drug Research Aims: new technologies for drug discovery and development new drugs and formulations Targets Molecules - synthesis - extracted - libraries Animal pharmacology &amp; toxicology Product development Processes Clinical phases Bioactivity screening Pharmacol. molecular biology Computational modeling eADME Preformulation Drug delivery &amp; targeting Analytical chemistry DDTC Slide 3 Drug Delivery and Targeting Group - Arto Urtti Main fields: 1) Nanosystems for Non-Viral Gene Delivery - mechanisms of gene delivery - new gene delivery systems and cell encapsulation fomulations - retinal pigment epithelium and neovessels as target cells 2) Ocular Drug Delivery - new cell models: RPE, corneal epithelium - ocular pharmacokinetic modeling - new polymeric delivery systems 3) ADME-methods for Drug Discovery and Development - cell model studies - QSAR and PK modeling Personnel: 1 senior scientist, 6 post-docs, 12 post-graduate students multidisciplinary Slide 4 PolExGene in Helsinki PolExGene kick off meeting Gent, August 23 and 24 2006 Arto Urtti Astrid Subrizi Slide 5 THE EYE RPE is between neural retina and choroid Blood retina barrier Slide 6 Role of University of Helsinki in PolExGene (Wp4,WP5) 1)Development of plasmids for polyplex incorporation 2)Funtionalisation of polymer membrane with polyplexes 3)Interaction between CPP containing polyplexes and cells 2) Interaction between CIP containing polymer membranes and cells Slide 7 Experiments in the Summer 2006: Methods ARPE-19 Human retinal pigment epithelia cell line Filter-culture cell model Dunn et al., Exp Eye Res. 1996;62:155169. Characterization of ARPE-19 Transfection with pCMV-SEAP Transepithelial Resistance (TER) Slide 8 Filter-culture cell model From E. Mannermaa et al. Curr Eye Res. 30:345353, 2005 Culture medium: DMEM-F12 (Gibco 31330-038) supplemented with 1% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/ml penicillin and 100 units/ml streptomycin. ARPE-19 cells were seeded at a density of 1.6 10 5 cells per cm 2 on laminin coated Transwell inserts (polycarbonate membrane, growth area 4.7 cm 2 ). Apical and basolateral media were routinely changed twice a week. Basolateral medium Apical medium ARPE- 19 cells DNA protein (SEAP) protein Slide 9 Differentiation markers Results of gene expression analysis using a singleplex TaqMan assay: Qualitative real-time PCR data show that both ARPE-19 cells grown on flask (for 3 weeks) and on filter, expressed the marker proteins CRALBP and RPE65. Threshold cycles (C T ) for flask-grown and filter-grown cells did not differ significantly. RNA isolation Reverse transcription (RT) RT-PCR experiment Data analysis CRALBP RPE65 RT-PCR Slide 10 Transfections with pCMV-SEAP Non-viral gene delivery was achieved by combining plasmid DNA with a cationic lipid (lipoplexes) or a cationic polymer (polyplexes). DOTAP/DOPE/PS:DNA (4,45:1) lipoplexes PEI:DNA (n/p 8 and 10) polyplexes Choice of the reporter gene: The SEAP gene product is secreted from transfected cells and is thus easily detected in a sample of culture medium, without destroying cells and allowing repeated culture sampling. Slide 11 Transepithelial Resistance (TER) To determine the time course and extent of tight junction formation, TER of ARPE-19 monolayers was recorded 2 weeks after seeding the cells, before transfection and at the end of the experiment. Transfection Slide 12 TASKS DURING 6 MONTHS Plasmid development 4.1. for basic characterisation CMV-SEAP, CMV-GFP EBNA - SEAP EBNA - PEDF, EBNA-CNTF Functionalisation of polymer membranes with polyplexes 4.4. CLSM method for visualisation of polyplexes embedded in nanofiber matrix Slide 13 TASKS DURING 6 MONTHS Interaction between polyplexes and cells 5.1. without CPP - basic data polyplexes and lipoplexes (see previous) cell uptake: FACS; tox: MTT; transfection duration of transfection: SEAP basic data : comparison EBNA plasmids vs non-relicating CPP containing plasmids when available Interaction between polymer membranes and cells 5.2. basic charcterisation differentiation, resistance, morphology (EM) Slide 14 Persons Involved Astrid Subrizi (Ph.D. student, biopharmacy) Eliisa Mannermaa (M.D., grad. student) Marjo Yliperttula (physical chemistry) Marika Hkli (molecular biology) Antti Laukkanen (polymer chemistry) Harri Palokangas (cell biology) Arto Urtti (biopharmaceutics) </p>

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