Date of Degree

6-2024

Document Type

Thesis

Degree Name

Master of Science (MS)

Program

Pharmaceutical Sciences

Advisor

Dr. Rheaclare Fraser-Spears

Advisor

Dr. Jorge Medina

Advisor

Dr. Georgiana Gould

Abstract

ABSTRACT

Organic cation transporter-3 (OCT3) is expressed abundantly throughout the body, but little is known about the presence of these transporters within the eye and, more specifically, the cornea epithelium. An extensive library of compounds interacts with OCT3, including the fluorescent molecule ASP+ and metformin. Metformin is an OCT3 substrate and antidiabetic medication that can lower the risk of eye-related diseases like keratopathy and retinopathy linked to hyperglycemia. Utilizing high throughput microplate assays in a human cornea epithelial cell line (HCE-S), the time-dependent saturation of ASP+ uptake and the competition of ASP+ uptake by corticosterone (CORT), a known selective inhibitor of OCT3 was evaluated. The hypothesis is that OCT3 is present and functional in human corneal epithelial HCE-S cells. The specific aims are to determine the functionality of OCT3 using saturation and competition assays that provide uptake capacity, transporter kinetics, and potency information. Saturation (Bmax and Kd) and competition (IC50) data help to establish the expression and function of OCT3 in HCE-S cells as a model to understand better the transporter’s function in the human cornea epithelial cells. The findings suggest OCT3 is robustly expressed in HCE-S cells and can function to transport ASP+. At baseline (25 mM glucose levels), the HCE-S cell line expresses transporters like OCT3 (and likely other isoforms) that can bind and transport the fluorescent substrate ASP+, which can be blocked by CORT and decynium22 (D22). The ASP+ saturation assay and time trial 30-minute uptake experiments are better for uptake capacity and affinity (Bmax ≅ 1100 RFU; Kd ≅ 272 µM). CORT competition assay using higher glucose levels may potentially decrease the potency of CORT to inhibit ASP+ uptake in HCE-S cells (IC50 ≅ 283 µM (baseline); ≅1717 µM (50 mM glucose, 24 hrs.). Our results also imply that other non-OCT3 transporters are present in this endogenous cell model capable of ASP+ uptake, as evidenced by our non-specific binding measurements using a non-selective inhibitor, D22. The next stage of competition experiments will use metformin to confirm that OCT3 is active in the HCE-S cell line. This work has future implications for establishing corneal cells as a new model to study OCT3 activity, test the pharmacological characteristics of ligands, and develop alternate administration routes for metformin (e.g., as eye drops).

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