ALTEX :: Alternatives to Animal Experiments

2001, VOLUME 1

Comparative Evaluation of in vitro Test Systems for Pulmonary Drug Delivery Based on Airway Epithelial Cell Lines and Human Alveolar Epithelial Cells (HAEC) in Primary Culture

Carsten Ehrhardt, Sabine Fuchs, Ulrich Friedrich Schäfer and Claus-Michael Lehr

Department of Biopharmaceutics and Pharmaceutical Technology, Saarland University, D-Saarbrücken

INTRODUCTION

Inhalation is an increasingly important route for drug delivery. Pharmaceutical aerosols are a well established means for either direct targeting to the lung, which results in an immediate onset of drug action and reduced side effects and furthermore the lung offers an elegant pathway for the systemic administration of orally non-deliverable drugs like peptides and proteins. Unfortunately there are no well developed in vitro test systems for the acquisition of drug permeability like for the gastro-intestinal tract.

PRIMARY CELLS

Human alveolar epithelial type II cells cultured on permeable filter supports (Transwell®) are able to differentiate to type I-like cells which form a confluent monolayer with transepithelial electrical resistance (TEER) values higher than 2000 cm2. The cells were derived from biopsies of bronchial adenocarcinoma. They were isolated as described earlier (Elbert et al., 1999) and cultured either at the air interface (AIC) or under liquid covered conditions. Since there are no published data about drug transport across the blood-air-barrier we tested the permeability of very lipophilic (verapamil) and very hydrophilic (FITC-dextran) marker compounds.

CELL LINES

Two cell lines have shown promise as in vitro absorption models of the airway epithelium: 16HBE14o- and Calu-3. They both form polarized monolayers and express the proteins of the mayor intercellular junctions (functional tight junctions, desmosomes and zonulae adherentes). 16HBE14o- cells, kindly donated by Dieter C. Gruenert, Cardiovascular Research Institute at the University of California, San Francisco, USA, are normal human airway epithelial cells, obtained from a one-year-old heart lung patient, transformed with SV40. The TEER values vary between 150 cm2 (AIC) and 600 cm2 (LCC). The Calu-3 cell line (ATCC HTB-55) was isolated from a bronchial adenocarcinoma in a 25-years-old male.

RESULTS

The permeability coefficient (Papp) of verapamil was found 1,895 ± 0.37 x 10-5 cm/sec compared to 8,75 ± 0.15 x 10-8 cm/sec for FD-4 dextran. Immunofluorescence of ZO-1 in HAEC and 16HBE14o- using a polyclonal antibody revealed clear expression of zonulae occludentes in both cell types and under both culture conditions (AIC and LCC). Caveolae which seem to be important in macromolecule trafficking and potocytosis are found in HAEC by TEM and their main structural protein caveolin was identified by western blotting.

CONCLUSION

The cellular in vitro models are a promising approach for the further study and characterisation of the airways and the blood-air-barrier. They allow the closer examination of drug transport processes as well as of metabolic activities.

ACKNOWLEDGEMENTS

The Heart Centre Völklingen (Dr. H. Isringhaus) and the Department of Thoracic Surgery, Saarland University, Homburg (Prof. Dr. H-J Schäfers) are being thanked for their regular supply with biopsy material. The ZEBET (BgVV, Berlin) is being thanked for the financial support.

REFERENCES

Elbert, K. et al. (1999). Monolayers of human alveolar epithelial cells in primary culture for pulmonary drug delivery and transport studies. Pharmaceutical Research 16, 601-609.