Melanin binding study of clinical drugs with cassette dosing and rapid equilibrium dialysis inserts

Abstract

Melanin pigment is a negatively charged polymer found in pigmented human tissues. In the eye, iris, ciliary body, choroid and retinal pigment epithelium (RPE) are heavily pigmented. Several drug molecules are known to bind to melanin, but larger sets of drugs have not been compared often in similar test conditions. In this study, we introduce a powerful tool for screening of melanin binding. The binding of a set of 34 compounds to isolated porcine RPE melanin was determined by cassette (n-in-one) dosing in rapid equilibrium dialysis inserts and the binding was quantitated with LC-MS/MS analytics. The compounds represented large variety in melanin binding (from 8.6%, ganciclovir) to over 95% bound (ampicillin and ciprofloxacin). The data provides information on melanin binding of small molecular weight compounds that are used for ocular (e.g. brinzolamide, ganciclovir) and systemic (e.g. tizanidine, indomethacin) therapy. Interestingly, competition among compounds was seen for melanin binding and the binding did not show any correlation with plasma protein binding. These results increase the understanding of melanin binding of ocular drugs and can be further exploited to predict pharmacokinetics in the eye. Pigment binding provides an interesting option for improved drug distribution to retina and choroid that are difficult target tissues in drug delivery.

RETRACTED: Drug Flux across RPE Cell Models: The Hunt for an Appropriate Outer Blood–Retinal Barrier Model for Use in Early Drug Discovery

“…This melanin content is similar to that present in intact porcine RPE [16]. The melanin amount was determined by an absorbance measurement (at 595 nm, Wallac Victor 2 1420 Multilabel counter, Perkin Elmer, MA, USA) utilizing isolated porcine RPE melanin standards (0.00-2.00 µg melanin/µL) [16,27]. Similar to the situation with regular ARPE19 cells, the ARPE19mel cells were cultured for 4 weeks before the experiments, in the same growth medium mentioned above.…”

“…The drug concentrations were determined with a liquid chromatograph (Agilent 1290, Agilent Technologies, Inc., Santa Clara, CA, USA) and a triple-quadrupole mass spectrometer (Agilent 6495; Agilent Technologies, Inc., Santa Clara, CA, USA) utilizing electrospray ionization in the positive ionization mode. We have previously described the method development and validation in detail [27,30], and a similar validation procedure was used in these LC-MS/MS runs.…”

Nanotechnology for Medical and Surgical Glaucoma Therapy—A Review

“…The trajectory of the drug molecules starts with their passive diffusion via the corneal epithelium, through the stroma and endothelium into the anterior chamber, where the drug will carry out its pharmacological action [28] or will bind to melanin in the ciliary Fig. 1 Glaucoma therapeutic strategies and nanotechnology-based potential applications body and iris [29], or plasma proteins [30], which prolongs its half-life.…”

Binding of Small Molecule Drugs to Porcine Vitreous Humor

“…In total 35 drugs were used in the study as a cassette mixture. The mixture was the same as used by Pelkonen et al 16 ( Table 1), and the drugs were dissolved at 1 mg/mL in DMSO or phosphate buffer as presented in their study. Before the study, the 1 mg/mL drug cassette was diluted to a concentration of 10 μg/mL with DPBS (pH 7.4, without MgCl 2 and CaCl 2 , Gibco, Thermo Fisher Scientific).…”

“…Cassette of Drugs Used in the Study a The mixture is the same as in Pelkonen et al16 Molecular Pharmaceutics…”