Outward-Directed Transport

Introduction

In ocular therapeutics, the current literature describes drug delivery limitations posed by outward directed transport mechanisms of the eye. Drug delivery to ocular tissues is restricted by various factors such as limited tissue penetration, rapid clearance rate, and dose-limiting toxicity. Though diffusion plays a role, efflux transport proteins constitute a major driving force behind selective removal of therapeutic agents from ocular tissues. Therapeutic agents must penetrate the globe through the anterior or posterior route, depending on the mode of administration. The cornea forms a major barrier for anterior segment drug delivery. Presence of epithelial tight junctions, along with a wide array of efflux transporters causes poor permeation of drugs across the cornea. Apart from these, the drug molecules should possess optimum physicochemical properties to penetrate the different corneal layers. The blood–retinal barrier (BRB) forms a major barrier to posterior segment ocular drug delivery. The BRB is composed of retinal vascular endothelium and retinal-pigmented epithelium and is categorized as inner BRB and outer BRB. Inner BRB is composed of a monolayer of endothelial cells, which are non-fenestrated, and outer BRB is composed of retinal-pigmented epithelial cells.

Multi-drug resistance mediated by the efflux transporter (pump) is a widely explored field in the area of cancer research. Efflux pumps belong to the ATP binding cassette (ABC) transporter family. Family members such as P-glycoprotein (P-gp), multidrug resistance associated protein (MRP), breast cancer resistance protein (BCRP) and lung resistance protein (LRP) are known to play a vital role in imparting drug resistance to cancer cells. Efflux transporters are known to be protective in preventing the entry of toxic substances into cells which results in outward directed transport of toxins.

The presence of efflux transporters on various physiological barriers protecting the eye was only recently discovered. Their existence in various ocular tissues such as cornea, retina, and conjunctiva has been established at molecular and functional level by various researchers. To a large extent the role of these efflux transporters in ocular drug resistance remains to be explored.

Various drug delivery strategies are employed to bypass the efflux transporters. The prodrug strategy, which involves linking of the active drug molecule to a promoiety, is known to reduce or eliminate the substrate specificity of drugs to the efflux transporters. Our recent findings revealed that amino acid and peptide prodrugs of quinidine, val-quinidine and val-val-quinidine were able to bypass the MRP class efflux transporters on the corneal epithelial cells. Earlier it was shown that val-quinidine and val-val-quinidine can effectively bypass P-gp mediated efflux on the cornea. This chapter comprises a discussion on the presence of efflux transporters in various ocular tissues and their possible role in outward directed transport. Various strategies employed to modulate or bypass efflux transporters are presented.

P-gp

P-gp in isolated primary culture of rabbit corneal epithelial cells was reported by Kawazu et al. The presence of P-gp at protein level and a series of uptake experiments demonstrated that the tear-side-facing membrane (apical) of rabbit corneal epithelial cells expressed P-gp. This article further concluded that the presence of P-gp on corneal cells protects them from toxic agents. Later the presence of P-gp in human cornea was demonstrated by Dey et al. This report further confirmed the expression of P-gp in rabbit cornea and rabbit corneal epithelial cells. Uptake of Rho 123, a P-gp substrate, was significantly higher in the presence of P-gp inhibitors like cyclosporine A (CsA) indicating the role of P-gp in corneal drug efflux. Ocular pharmacokinetic studies involving male New Zealand Albino rabbits revealed a significant elevation in aqueous humor drug concentrations in the presence of various P-gp inhibitors, i.e. testosterone, quinidine, verapamil and CsA. Moreover dose-dependent suppression in P-gp efflux was evident at various inhibitor concentrations. The presence of P-gp in the cornea was corroborated by a recent report on the quantitative expression of P-gp in human cornea. The relative role of P-gp in the outward directed efflux of drugs used in glaucoma, anti-bacterial and anti-viral agents has yet to be established.

An ocular microdialysis technique was applied to determine the role of efflux transporters on the cornea. Figure 17.1 shows a schematic diagram of the surgical implantation of the microdialysis probe. A well is placed on the top of cornea filled with drug solution. The sample is collected from the anterior end of the probe which protrudes from the cornea. Drug diffuses across the dialyzing membrane placed in the aqueous humor. This technique helps to quantitatively determine the drug concentrations in the aqueous humor at different time points. This study reported that P-gp inhibitors significantly raised drug concentration in aqueous humor (both C _max and AUC) ( Table 17.1 ). The results demonstrated that P-gp can play a vital role in conferring resistance to drug absorption across the cornea. This article also suggested the use of efflux pump inhibitors along with therapeutic agents – a strategy that helps to increase the overall ocular bioavailability of drugs which are substrates of efflux pumps.

Along with cornea, the BRB limits the permeability of various therapeutic agents into the eye. Retinal pigmented epithelium (RPE), situated between the retina and its choroidal blood supply, forms a diffusional barrier controlling access to subretinal space. Transport proteins present on the RPE regulate ionic composition and hydration of the subretinal space. Immunohistochemistry revealed that P-gp is localized both on the apical and basolateral sides of human RPE. Basolateral P-gp is attributed to the function of neural retina in clearing toxic and unwanted molecules from the subretinal space. Apical expression of P-gp appears to assist in the delivery of lipophilic substrates to subretinal space and engulfment by photoreceptors. Three RPE cell lines, ARPE-19, D-407 and h1RPE, are usually employed for in vitro studies. Screening of these three cell lines for P-gp expression revealed that D-407 is suitable for functional expression and drug transport screening. ARPE-19 cells lacked both molecular and functional expression of P-gp, whereas h1RPE lacked functional expression of P-gp. D-407 demonstrated both molecular and functional expression of P-gp. Also, P-gp was found to be expressed on the choroidal blood vessels. Further work is in progress to understand the role of P-gp in the choroid.

P-gp is also expressed in rabbit conjunctival epithelial cells. It is present on the apical side of the conjunctiva, conferring resistance to the absorption of cyclosporine A and other lipophilic drugs. Use of various P-gp inhibitors – such as verapamil, progesterone, cyclosporine A, rhodamine 123 and metabolic inhibitor 2,4-DNP – enhanced the uptake of the model drug propranolol in rabbit conjunctival epithelial cells. Yang et al concluded that P-gp can play a vital role in decreased absorption of various lipophilic drugs across the conjunctiva.

P-gp appears to play an important role in the outward transport of cytotoxic drugs and in the modulation of chloride channels which regulate and initiate osmotic cataract. Further, findings by Miyazawa et al revealed that P-gp might be induced by aldose reductase overexpression and osmotic stress. P-gp was found to maintain lenticular osmotic balance in sugar cataract. The role of P-gp in the drug disposition of various ocular agents is being further evaluated.

Mode of action and structure of P-gp

P-gp requires ATP for its function. There are two ATP binding sites, also known as nucleotide binding domains (NBD). Apart from NBD, there are two transmembrane domains incorporating six transmembrane domains at each site (a total of 12). A pictorial representation of P-gp is provided in Figure 17.2 .