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Drug Discovery and Development Technology Center

Drug Discovery and Development Technology Center. Independent Research Centre in the Faculty of Pharmacy Multidisciplinary Networking: Faculty, other Funding. Early-ADME &preformulation. Drug Discovery. Pharmaceutical Nanotechnology. DDTC in Drug Research. Aims :

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Drug Discovery and Development Technology Center

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  1. Drug Discovery and Development Technology Center Independent Research Centre in the Faculty of Pharmacy Multidisciplinary Networking: Faculty, other Funding Early-ADME &preformulation Drug Discovery Pharmaceutical Nanotechnology

  2. DDTC in Drug Research • Aims: • new technologies for drug discovery and development • new drugs and formulations DDTC Animal pharmacology & toxicology Product development Processes Clinical phases • Bioactivity screening • Pharmacol. molecular • biology • Computational modeling • eADME • Preformulation • Drug delivery & targeting • Analytical chemistry • Targets • Molecules • synthesis • extracted • libraries

  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

  4. PolExGene in Helsinki PolExGene kick off meeting Gent, August 23 and 24 2006 Arto Urtti Astrid Subrizi

  5. THE EYE RPE is between neural retina and choroid Blood retina barrier

  6. Role of University of Helsinki in PolExGene (Wp4,WP5) Development of plasmids for polyplex incorporation Funtionalisation of polymer membrane with polyplexes Interaction between CPP containing polyplexes and cells 2) Interaction between CIP containing polymer membranes and cells

  7. Experiments in the Summer 2006: Methods

  8. Apical medium DNA ARPE-19 cells protein protein (SEAP) Filter-culture cell model From E. Mannermaa et al. Curr Eye Res. 30:345–353, 2005 Basolateral medium • 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 × 105 cells per cm2 on laminin coated Transwell inserts (polycarbonate membrane, growth area 4.7 cm2). • Apical and basolateral media were routinely changed twice a week.

  9. Differentiation markers RNA isolation Reverse transcription (RT) RT-PCR experiment Data analysis CRALBP RPE65 RT-PCR • 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 (CT) for flask-grown and filter-grown cells did not differ significantly.

  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.

  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 Transfection

  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

  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)

  14. Persons Involved • Astrid Subrizi (Ph.D. student, biopharmacy) • Eliisa Mannermaa (M.D., grad. student) • Marjo Yliperttula (physical chemistry) • Marika Häkli (molecular biology) • Antti Laukkanen (polymer chemistry) • Harri Palokangas (cell biology) • Arto Urtti (biopharmaceutics)

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