title: Interpretation of Ocular Melanin Drug Binding Assays. Alternatives to the Model of Multiple Classes of Independent Sites.
abstract: Melanin has a high binding affinity for a wide range of drugs. The determination of the melanin binding capacity and its binding affinity are important, e.g., in the determination of the ocular drug distribution, the prediction of drug effects in the eye, and the trans-scleral drug delivery. The binding parameters estimated from a given data set vary significantly when using different isotherms or different nonlinear fitting methods. In this work, the commonly used bi-Langmuir isotherm, which assumes two classes of independent sites, is confronted with the Sips isotherm. Direct, log-log, and Scatchard plots are used, and the interpretation of the binding curves in the latter is critically analyzed. In addition to the goodness of fit, the emphasis is placed on the physical meaning of the binding parameters. The bi-Langmuir model imposes a bimodal distribution of binding energies for the sites on the melanin granules, but the actual distribution is most likely continuous and unimodal, as assumed by the Sips isotherm. Hence, the latter describes more accurately the distribution of binding energies and also the experimental results of melanin binding to drugs and metal ions. Simulations are used to show that the existence of two classes of sites cannot be confirmed on the sole basis of the shape of the binding curve in the Scatchard plot, and that serious doubts may appear on the meaning of the binding parameters of the bi-Langmuir model. Experimental results of melanin binding to chloroquine and metoprolol are used to illustrate the importance of the choice of the binding isotherm and of the method used to evaluate the binding parameters.
authors.affiliation: Department of Thermodynamics, Faculty of Physics, University of Valencia , E-46100 Burjassot, Spain.
authors.forename: José A
authors.initials: JA
authors.lastname: Manzanares
authors.affiliation: Centre for Drug Research, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki , P.O. Box 56, 00014 Helsinki, Finland.
authors.forename: Anna-Kaisa
authors.initials: AK
authors.lastname: Rimpelä
authors.affiliation: Centre for Drug Research, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki , P.O. Box 56, 00014 Helsinki, Finland.|School of Pharmacy, University of Eastern Finland , P.O. Box 1627, 70211 Kuopio, Finland.
authors.forename: Arto
authors.initials: A
authors.lastname: Urtti
mesh_terms: D002738:Chloroquine / Q000737:chemistry; D007700:Kinetics; D008543:Melanins / Q000737:chemistry*; D008790:Metoprolol / Q000737:chemistry; D008962:Models, Theoretical
chemical_list: D008543:Melanins; D002738:Chloroquine; D008790:Metoprolol
keywords: Langmuir−Freundlich isotherm; Scatchard plot; Sips isotherm; bi-Langmuir isotherm; chloroquine; drug binding assays; metoprolol; multiple classes of independent sites; ocular melanin; site energy distribution function