School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, 70210, Kuopio, Finland.
Drug Research Programme, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, FI-00014, Helsinki, Finland.
Sci Rep. 2019 Sep 24;9(1):13761. doi: 10.1038/s41598-019-50324-8.
Melanin pigment has a significant role in ocular pharmacokinetics, because many drugs bind at high extent to melanin in the retinal pigment epithelial cells. Most retinal pigment epithelial cell lines lack pigmentation and, therefore, we re-pigmented human ARPE-19 cells to generate a pigmented cell model. Melanosomes from porcine retinal pigment epithelium were isolated and co-incubated with ARPE-19 cells that spontaneously phagocytosed the melanosomes. Internalized melanosomes were functionally integrated to the cellular system as evidenced by correct translocation of cellular Rab27a protein to the melanosomal membranes. The pigmentation was retained during cell cultivation and the level of pigmentation can be controlled by altering the amount of administered melanosomes. We used these cells to study melanosomal uptake of six drugs. The uptake was negligible with low melanin-binders (methotrexate, diclofenac) whereas most of the high melanin-binders (propranolol, chloroquine) were extensively taken up by the melanosomes. This cell line can be used to model pigmentation of the retinal pigment epithelium, while maintaining the beneficial cell line characteristics, such as fast generation of cultures, low cost, long-term maintenance and good reproducibility. The model enables studies at normal and decreased levels of pigmentation to model different retinal conditions.
黑色素在眼部药代动力学中具有重要作用，因为许多药物与视网膜色素上皮细胞中的黑色素高度结合。大多数视网膜色素上皮细胞系缺乏色素，因此，我们重新给人 ARPE-19 细胞着色，以生成一个着色细胞模型。从猪的视网膜色素上皮中分离出黑素小体，并与 ARPE-19 细胞共孵育，这些细胞会自发吞噬黑素小体。内吞的黑素小体被功能性地整合到细胞系统中，这可以通过细胞 Rab27a 蛋白正确地转移到黑素小体膜上得到证明。在细胞培养过程中保留了色素沉着，并且可以通过改变给予的黑素小体的量来控制色素沉着的水平。我们使用这些细胞来研究黑素小体对六种药物的摄取。低黑色素结合物（甲氨蝶呤、双氯芬酸）的摄取可以忽略不计，而大多数高黑色素结合物（普萘洛尔、氯喹）则被黑素小体大量摄取。该细胞系可用于模拟视网膜色素上皮的色素沉着，同时保持有益的细胞系特征，如培养物的快速生成、低成本、长期维持和良好的重现性。该模型可用于模拟不同的视网膜条件下正常和减少的色素沉着水平。
