Cellular pharmacokinetic and pharmacodynamic analyses of ethacrynic acid: Implications in topical drug delivery in the eye

Abstract

Purpose: Ethacrynic acid (ECA) is a potential trabecular meshwork (TM) drug that has shown promising results in preclinical studies for treatment of primary open-angle glaucoma. However, topical application of ECA is currently limited by adverse effects in corneal tissues. To this end, we developed a new theoretical model to evaluate time-dependent toxicity induced by ECA in corneal epithelial cells.

Methods: The model consisted of a cellular pharmacokinetic (PK) module to determine intracellular concentration of ECA, and a pharmacodynamic (PD) module to determine the cytotoxicity of ECA. It was assumed that ECA-induced cytotoxicity depended on drug exposure time and peak concentration of bound ECA in cells. In addition to the model development, we experimentally determined the intracellular concentration of ECA as a function of drug dose and treatment time.

Results: The intracellular concentration increased linearly (i.e., no saturation) with increasing the dose of ECA. It also increased initially with time and then reached a steady-state at ~40 min. The percent of cells survived after treatment decreased with increasing the dose of drug or the time of treatment. The experimental data were fit by the new PK and PD models to obtain values of model constants. One of the unique applications of these models was to predict cell survival relative to control when extracellular concentration of ECA varied with time. The prediction showed that the toxicity of ECA might be significantly overestimated by using the traditional LC(50) determined in vitro.

Conclusions: The new PK and PD models developed in this study were capable to fit experimental data and predict time-dependent toxicity of ECA in corneal epithelial cells. The models may be useful for optimizing the dose and schedule in topical application of ECA for glaucoma treatment.

Figure 1

UV absorbance spectrums of ECA and GSH. The concentrations of ECA and GSH were 50 μM and 600 μM, respectively. The spectrum curves suggested that ECA concentration should be measured at 270 nm to minimize possible interference with signals from GSH.

Figure 2

Schematic representation of the proposed pharmacokinetic model. The definition of each term is as follows: C_e: extracellular ECA concentration; C_if: intracellular concentration of free (unbound) ECA; G: intracellular binding site available to ECA; G-ECA: bound ECA; C_ib: intracellular concentration of bound ECA; and P_m: permeability coefficient of cell membrane.

Figure 3

Intracellular concentration of ECA. It was a function of the initial extracellular concentration of ECA and the period of treatment.

Figure 4

Time dependent changes in intracellular concentration of ECA. The symbols indicate experimental data and the curves were resulted from fitting the PK model to the data. The best-fit values of the model constants are reported in Table 1.

Figure 5

Experimental data of cell survival relative to control (S). It was a function of the initial extracellular concentration of ECA and the period of treatment. The symbols indicate experimental data and the curves were resulted from fitting the PD model to the data. ●: 2 h of constant ECA exposure; ▲: 6 h of constant ECA exposure; ◆: 24 h of constant ECA exposure; ◇: 72 h of ECA exposure. The best-fit values of the model constants are reported in Table 1.

Figure 6

Cell survival relative to control simulated by using the cellular PK and PD models. S was plotted as a function of the initial concentration of ECA in the extracellular medium for three different scenarios of topical drug delivery in the eye. (i) ECA was applied via traditional eye drops (LC₅₀=2330 μM); (ii) ECA was released from a Pluronic^® copolymer film (LC₅₀=540 μM); and (iii) ECA concentration at corneal surface was maintained at constant levels for 1,000 h (LC₅₀=35 μM).