P-glycoprotein Restricts Ocular Penetration of Loperamide across the Blood-Ocular Barriers: a Comparative Study in Mdr1a Knock-out and Wild Type Sprague Dawley Rats

Abstract

The current research was undertaken to determine the existence and magnitude of P-glycoprotein (P-gp) expression on the blood-ocular barriers by studying the ocular penetration of loperamide, a specific P-gp substrate, in P-gp (Mdr1a) knock-out (KO) and wild type (WT) Sprague Dawley rats. A clear, stable, sterile solution of loperamide (1 mg/mL), for intravenous administration, was formulated and evaluated. Ocular distribution was studied in P-gp KO and WT rats following intravenous administration of loperamide (at two doses). The drug levels in plasma, aqueous humor (AH), and vitreous humor (VH) samples were determined with the aid of UHPLC-Q-TOF-MS/MS, and the AH/plasma (D _AH ) and VH/plasma (D _VH ) distribution ratios were estimated. Electroretinography (ERG), ultrastructural analyses, and histology studies were carried out, in both KO and WT rats, to detect any drug-induced functional and/or structural alterations in the retina. Dose-related loperamide levels were observed in the plasma of both WT and KO rats. The loperamide concentrations in the AH and VH of KO rats were significantly higher compared to that observed in the WT rats, at the lower dose. However, a marked increase in the D _AH and D _VH was noted in the KO rats. ERG, ultrastructure, and histology studies did not indicate any drug-induced toxic effects in the retina under the test conditions. The results from these studies demonstrate that P-gp blocks the penetration of loperamide into the ocular tissues from the systemic circulation and that the effect is more pronounced at lower plasma loperamide concentrations.

Keywords: P-glycoprotein; blood-ocular barriers; electroretinography; ocular distribution; retinal pigmented epithelium.

Fig. 1.

Plasma concentration of loperamide in WT and P-gp KO rats, (mean ± SEM; n=6 rats). *-indicates a significant difference (p<0.05) in plasma concentrations between WT 0.5 mg/kg and WT 1 mg/kg, or between KO 0.5 mg/kg and KO 1 mg/kg, at the same time point.

Fig. 2.

Aqueous humor (AH) concentration of loperamide in WT and P-gp KO rats, (mean ± SEM; n=6 rats). N.D.- not detected; *- indicates a significant difference (p<0.05) in AH concentrations between WT 0.5 mg/kg and KO 0.5 mg/kg, at the 120 min point; ǂ - indicates a significant difference (p<0.05) in AH concentrations between KO 0.5 mg/kg at 120 min and KO 0.5 mg/kg, at 60 min point or between KO 0.5 mg/kg at 120 min and KO 1 mg/kg, at 60 or 120 min.

Figure 3.

Vitreous humor (VH) concentration of loperamide in WT and P-gp KO rats, (mean ± SEM; n=6 rats). N.D.-not detected; < LOQ – below the LOQ; *- indicates a significant difference (p<0.05) between the KO 0.5 mg/kg vs WT 0.5 mg/kg, at 120 min time point. ǂ - indicates a significant difference (p<0.05) between the KO 0.5 mg/kg (120 min) vs KO 0.5 mg/kg (60 min), or between the KO 0.5 mg/kg (120 min) vs KO 1 mg/kg (60 min or 120 min).

Figure 4.

Electroretinography (ERG) data showing percentage changes of a- and b-wave amplitudes, from baseline, in WT and P-gp KO rats at A) 2h, B) 1day and C) 3days after intravenous administration of loperamide (low dose), (mean ± SEM; n=3). * - indicates significantly different from baseline (p<0.05).

Figure 5.

Retinal morphology of WT and P-gp KO rats 2h post intravenous administration of 0.5 mg/kg loperamide. A) and B) are the histology images illustrating a transverse section of retina from WT and KO rats, respectively. C) and D) are the TEM images of the RPE in WT and KO rats, respectively. Ch-choriocapillaris; RPE-retinal pigmented epithelium; OS-outer segment; IS-inner segment; ONL-outer nuclear layer; INL-inner nuclear layer; BM-Bruch’s membrane; PG-pigmented granules.