An Investigation into Retigabine (Ezogabine) Associated Dyspigmentation in Rat Eyes by MALDI Imaging Mass Spectrometry

Retigabine (RTG) is an antiepileptic drug approved as an adjunctive treatment for refractory partial-onset seizures in adults. In April 2013, the Food and Drug Administration issued a warning that RTG could cause changes in retinal pigmentation and discoloration of skin, resulting in a blue appearance. As part of a larger preclinical effort to gain a mechanistic understanding as to the origins of retinal pigment changes associated with RTG, we conducted a long-term repeat dosing study in rats. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) was used to determine the distribution of RTG and its metabolites in the rat eye following 13 and 39 weeks of dosing. IMS revealed the presence of RTG, a previously characterized N-acetyl metabolite of RTG (NAMR), and several species structurally related through the dimerization of RTG and NAMR. These species were highly localized to the melanin-containing layers of the uveal tract of the rat eye including the choroid, ciliary body, and iris, suggesting that the formation of these dimers occurs from melanin bound RTG and NAMR. Furthermore, several of the RTG-related dimers have UV absorbance which give them a purple color in solution. We propose that the melanin binding of RTG and NAMR effectively concentrates the two compounds to enable mixed condensation reactions to occur when the binding provides the proper geometry in the redox environment of the uveal tissues. High lateral resolution images illustrate that the blood-retinal barrier effectively restricts retinal access to RTG-related compounds. The spatial information provided by MALDI IMS was critical in contextualizing the homogenate concentrations of key RTG-related compounds and helped provide a basis for the mechanism of dimer formation.