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Silvia Pescina, Patrizia Santi, Giulio Ferrari, Sara Nicoli; In-vitro Model To Study The Role Of Melanin In Transscleral Drug Delivery. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3226.
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To set up an in-vitro model to study the role of melanin as a barrier in transscleral drug delivery.
Brown and light blue porcine eye bulbs (taken from a local slaughterhouse) were dissected within two hours from animal death: vitreous, retina and RPE were removed and the bilayer choroid-Bruch’s membrane was isolated from the sclera and mounted on vertical diffusion cells. Brown eyes, due to the presence of melanin, are characterized by a black choroid, while light blue eyes show a not pigmented bilayer. Permeation experiments were performed through pigmented and not pigmented bilayers using as model compounds methylene blue, levofloxacin (both have high affinity for melanin) and a high molecular weight (39KDa) linear dextran. Additionally, melanin binding curves of methylene blue and levofloxacin were built using both melanin from Sepia officinalis and melanin enzimatically extracted from porcine eyes.
Permeation profiles through pigmented and not pigmented bilayers resulted superimposable in the case of the linear dextran, a hydrophilic and uncharged molecule that does not show any affinity for melanin. Methylene blue, on the contrary has high affinity for melanin as demonstrated through in-vitro binding studies. When this molecule was applied to the not pigmented bilayer (conc. 100 µg/ml) the flux obtained was 15.6±3.4 µg/cm2h, while using the pigmented bilayer no permeation took place even after 2 hours. Increasing the concentration to 500 µg/ml the flux was identical through pigmented and not pigmented tissue, probably due to the saturation of melanin binding sites; however, the time lag, that was approximately 10 minutes in the case of not pigmented bilayer, increased to 55 minutes in the presence of melanin. In-vitro binding studies confirmed the levofloxacin-melanin interaction, even if the affinity resulted from 3 (melanin from Sepia officinalis) to 40 (melanin from porcine eyes) fold lower than in case of methylene blue. Permeation profiles through pigmented and not-pigmented tissues were superimposable indicating that, due to the lower affinity, the concentrations used are able to saturate melanin binding sites for levofloxacin.
The in-vitro model set up represents an easy method for the evaluation of the barrier role of melanin in the transscleral delivery of drugs. Compared to melanin binding studies, this in-vitro model can give more appropriate information on saturation concentration and could also help to understand the role of melanin on lag time in transscleral permeation.
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