Upload
omari-horwich
View
227
Download
0
Tags:
Embed Size (px)
Citation preview
Drug Discovery and Development Technology Center
Drug Discovery
Early-ADME&preformulation
Pharmaceutical Nanotechnology
Independent Research Centre in the Faculty of Pharmacy Multidisciplinary
Networking: Faculty, other
Funding
DDTC in Drug Research• Aims:
– new technologies for drug discovery and development
– new drugs and formulations
Targets
Molecules- synthesis- extracted- libraries
Animalpharmacology& toxicology
Product development
Processes
Clinical phases
• Bioactivity screening•Pharmacol. molecular
biology•Computational modeling
• eADME• Preformulation
• Drug delivery & targeting• Analytical chemistry
DDTC
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 studentsmultidisciplinary
PolExGene in Helsinki
PolExGene kick off meetingGent, August 23 and 24 2006
Arto UrttiAstrid Subrizi
THE EYE
RPE is between neuralretina and choroid
Blood retinabarrier
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
Experiments in the Summer 2006: Methods
ARPE-19Human retinal pigment
epithelia cell line
Filter-culturecell model
Dunn et al., Exp Eye Res. 1996;62:155–169.
Transfection withpCMV-SEAP
Transepithelial Resistance (TER)
Characterization of ARPE-19
Filter-culture cell modelFrom E. Mannermaa et al.Curr Eye Res. 30:345–353, 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 × 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.
Basolateral medium
Apical medium
ARPE-19 cells
DNA
protein (SEAP)
protein
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 (CT) 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
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) lipoplexesPEI: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.
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.
PEI/DNA complexes
40
45
50
55
60
65
70
10 20 30 40 50
Days after seeding
Resistance [_/cm2]
DOTAP/DOPE/PS/DNA complexes
40
45
50
55
60
65
70
10 20 30 40 50
Days after seeding
Resistance [_/cm2]
Transfection Transfection
TASKS DURING 6 MONTHSPlasmid 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
TASKS DURING 6 MONTHSInteraction 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)
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)