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Anna-Kaisa Rimpelä, Mechthild Schmitt, Satu Latonen, Marja Hagström, Maxim Antopolsky, José Antonio Manzanares, Heidi Kidron, Arto Urtti; Drug distribution to retinal pigment epithelium: studies on melanin binding, cellular kinetics and SPECT/CT imaging. Invest. Ophthalmol. Vis. Sci. 2016;57(12):107.
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© ARVO (1962-2015); The Authors (2016-present)
The effect of melanin binding on drug distribution to the retinal pigment epithelium (RPE) is inadequately understood. We evaluated if the extent of drug uptake to primary retinal pigment epithelial (RPE) cells could be estimated based on in vitro binding studies with isolated melanin and if single photon emission computed tomography/computed tomography (SPECT/CT) imaging could be used in studying pigment binding in vivo with pigmented and albino rats.
Melanin binding of three basic compounds (timolol, nadolol and chloroquine) and two acidic compounds (methotrexate and 5(6)-carboxy-2',7'-dichlorofluorescein (CDCF)) was studied with isolated melanin from porcine RPE-choroid by incubating the compounds at concentrations 0.25-500 µM in 1 mg/ml melanin suspension at pH 5.0 (citrate buffer) and pH 7.4 (phosphate buffer). Cellular uptake and elimination of timolol, chloroquine, methotrexate and CDCF was studied in primary porcine RPE cells. The concentration of the compounds in samples taken from the medium was analyzed by chromatographic and mass spectrometric methods. Cellular uptake was predicted based on the experiments with isolated melanin. Ocular retention of systemically administered 123I-chloroquine was studied for 24 h with SPECT/CT imaging in pigmented (N=2) and albino rats (N=2).
The basic compounds bound to melanin at both pH values (Figure 1). The acidic compounds did not bind considerably at pH 7.4 but had significant binding at pH 5.0. The basic compounds (timolol, chloroquine) showed significant cellular uptake to primary RPE cells, unlike the acidic compounds (methotrexate, CDCF). According to the predictions made based on results with isolated melanin at pH 7.4, melanin binding was a major factor governing the overall drug distribution to the cells. 123I-chloroquine was retained in the eyes of the pigmented rats but not the albino rats until the end of the experiment (24 h).
Drug distribution to the RPE is governed by melanin binding and can be evaluated based on in vitro binding experiments with isolated melanin. Ocular melanin binding can be monitored in vivo with non-invasive SPECT/CT imaging.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
Figure 1. The range of binding of the studied compounds to isolated melanin at pH 5.0 and 7.4 with starting concentrations 0.25-100 µM (nadolol pH 7.4 0.5-100 µM). Error bars indicate standard deviation.
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