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)