TY  - THES
T1  - Examination of the penetration behavior of pharmaceutical drugs on the eye and set-up of an in virto cell model for ocular drug delivery
A1  - Becker,Ulrich
Y1  - 2007/01/25
N2  - The focus of this thesis was to find a simple test system to screen new therapeutic entities to regarding their ocular penetration. Using the model compound moxaverine-hydrochloride, the project was performed in 4 phases:

In phase 1, the tissue distribution of moxaverine-hydrochloride in the rabbit eye was examined and evaluated. A distribution profile was generated by either dosing pigmented Dutch-belted rabbits topically on the eye or applying the drug solution intravenously. Results showed that high drug amounts in the posterior part of the eye can be achieved after pre-corneal disposition of the moxaverine-solution. The fact that blood-plasma levels of moxaverine-hydrochloride did not exceed the levels found after systemic dosing makes local ocular administration of moxaverine-hydrochloride an interesting therapeutic approach.
Parallel to the in vivo testing, the barrier that corneal epithelium exhibits towards moxaverine-hydrochloride was examined using a primary rabbit corneal epithelial cell culture (rbCECL). These in vitro drug transport studies confirmed that the rabbit cornea does not represent a critical barrier for moxaverine-penetration into the eye.

Phase 2 of the presented project dealt with the development of a primary human corneal epithelial cell model. 
The methods mainly concentrated on various enzymatic digestions, using proteases and combination of these enzymatic techniques with a cell positive selection protocol using magnetic microbeads coated with human epithelial antigen (CD327, HEA-125). Even though the experimental conditions were varied intensively and numerous combinations of enzymes were examined, the aim of the primary human corneal epithelial cell culture was not reached. The limited amount of available tissue and the long storage time of the corneal samples after excision might contribute to the failure of this part of the work.

Since a primary human corneal epithelium was out of reach as an in vitro test system, existing models of human corneal epithelium were examined and compared to intact human and rabbit cornea (phase 3 of the thesis). The cell models under investigation were all of human origin and immortalized by either SV-40 large T antigen or a recombinant retrovirus containing HPV16 genes E6 and E7. The cell models were examined by a spectrum of marker-substances for their power to differentiate (high/low permeability markers), presence and activity of P-glycoprotein efflux systems (using substrate molecules), and value for ophthalmic research ("ophthalmology marker';). The systems were also used to screen the substance of interest, moxaverine-hydrochloride.
Using a set of marker substances, a ranking of the models was created. The commercially available reconstituted human corneal epithelium from Skinethic (Nice, France) showed a poor performance in this study, even though it serves as a valuable tool in toxicity and eye irritation testing. The likewise commercially available Clonetics system from Cambrex as well as the long established HCE-T cell line proved to be good simplified models of the cornea. Drawbacks for the HCE-T cell line, however, is the less pronounced power of differentiation and the findings made in stage 4 of the project (see below).

In the final, fourth phase of the project, the HCE-T cell line was compared to the human cornea regarding its equipment with common efflux transporters. Since efflux systems of the ABC-transporter family are known to be a crucial factor in drug disposition and failure in cancer therapy, these proteins have gained major interest in biopharmaceutical research. 
To clarify the situation in corneal epithelium that has not been extensively reviewed so far, we examined the presence of MDR1/P-gp, MRP1, MRP2, LRP, and BCRP in ex vivo human corneal samples and the commonly used HCE-T cell line. RT-PCR and immunofluorescence microscopy, as well as immunoblotting were used as tools to assess the spectrum of transporters.
Human cornea proved to be a rather uncritical tissue concerning multi-drug resistance issues, showing only a little array of efflux proteins, namely only LRP, HCE-T, however, showed a broader spectrum of efflux proteins, not unexpected in a continuously growing cell line that highly differs from the situation found ex vivo.
KW  - Penetration
KW  - Auge
KW  - Hornhaut
KW  - Arzneimittel
KW  - Zellkultur
KW  - Vergleich
KW  - Multidrug-Resistenz
KW  - In vitro
CY  - Saarbrücken
PB  - Universitäts- und Landesbibliothek
AD  - Postfach 151141, 66041 Saarbrücken
UR  - http://scidok.sulb.uni-saarland.de/volltexte/2007/946
ER  -