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Binding of Betaxolol, Metoprolol and Oligonucleotides to Synthetic and Bovine Ocular Melanin, and Prediction of Drug Binding to Melanin in Human Choroid-Retinal Pigment Epithelium

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Abstract

Purpose

To characterize the binding of betaxolol, metoprolol and oligonucleotides to synthetic and bovine ocular melanin, and to predict the binding to melanin in human choroid-retinal pigment epithelium (RPE).

Materials and Methods

The shape, size and specific surface area of synthetic melanin and isolated melanin granules from bovine choroid-retinal pigment epithelium (RPE) were characterized by SEM, laser diffractometry and BET. The binding of betaxolol, metoprolol, fluorescein isothiocyanate (FITC)-labeled phosphodiesther oligonucleotides and 6-carboxyfluorescein (6-CF) to melanin was determined. The binding of beta-blockers to melanin in human choroid-RPE was estimated based on binding parameters and the melanin content in human choroid-RPE.

Results

Bovine melanin granules were round or oval with a mean diameter of ca. 1 μm. Synthetic granules were slightly smaller and irregular and had a two times higher specific surface area than bovine melanin. Synthetic melanin bound more betaxolol and metoprolol than bovine melanin and both melanin types showed a high affinity and a low affinity binding sites. The human choroid-RPE was predicted to contain 3–19 times more melanin bound drug than unbound drug at typical therapeutic concentrations (1–1,000 ng/ml). FITC-labeled oligonucleotides and 6-CF did not bind to melanin.

Conclusions

The binding of lipophilic drugs to biological melanin differs from that of synthetic melanin. Lipophilic beta-blockers are expected to bind significantly to melanin in human choroid-RPE: only a small fraction of the drug being in active free form. In contrast, phosphodiesther oligonucleotides do not seem to bind to melanin.

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Abbreviations

BET:

Brunauer, Emmet, Teller-method

CF:

carboxyfluorescein

DHIC:

dihydroxyindole-2-carboxylic acid

FITC:

fluorescein isothiocyanate

HEPES:

N-2-hydroxyethylpiperazine-N-′2-ethane sulphonic acid

HPLC:

high performance liquid chromatography

PB8:

potassium phosphate buffer, pH 8

RPE:

retinal pigment epithelium

SEM:

scanning electron microscopy

TFA:

trifluoroacetic acid

VEGF:

vascular endothelial growth factor

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Acknowledgements

The authors acknowledge financial support from the Academy of Finland, the National Agency of Technology and the Farmos Research and Science Foundation.

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Authors and Affiliations

  1. Department of Pharmaceutics, University of Kuopio, P.O. Box 1627, Kuopio, 70211, Finland

    Leena Pitkänen, Veli-Pekka Ranta, Hanna Moilanen & Arto Urtti

  2. Department of Ophthalmology, Kuopio University Hospital, P.O.Box 1777, Kuopio, 70211, Finland

    Leena Pitkänen

  3. Drug Discovery and Development Technology Center, University of Helsinki, Helsinki, Finland

    Arto Urtti

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  1. Leena Pitkänen
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  2. Veli-Pekka Ranta
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Correspondence to Leena Pitkänen.

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Pitkänen, L., Ranta, VP., Moilanen, H. et al. Binding of Betaxolol, Metoprolol and Oligonucleotides to Synthetic and Bovine Ocular Melanin, and Prediction of Drug Binding to Melanin in Human Choroid-Retinal Pigment Epithelium. Pharm Res 24, 2063–2070 (2007). https://doi.org/10.1007/s11095-007-9342-0

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  • DOI: https://doi.org/10.1007/s11095-007-9342-0

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