4.1  Introduction

Retinal diseases such as age-related macular degeneration, diabetic retinopathy, and glaucoma have become an important therapeutic target with urgent medical needs. Although the ophthalmic drug market is dominated by topical eye drop formulations for anterior segment drug therapies (Del Amo and Urtti 2008), development of systemic drug delivery to the retina poses various hurdles in the treatment of retinal diseases. In general, the restricted drug penetration rate from the circulating blood to the retina is a major problem for retinal drug therapies. The retina is protected by the blood-retinal barrier (BRB; Fig. 4.1) from potentially harmful compounds that are present in the systemic circulation and produced in the retina. Although this role of the BRB is certainly beneficial to the retina, it also reduces the efficacy in the retinal drug delivery via systemic administration. However, it has become increasingly clear in recent years that the BRB performs the vectorial transfer of nutrients in the blood-to-retina direction and also eliminates metabolic waste products in the retina- to-blood direction (Hosoya and Tachikawa 2009). Such information would be useful

Fig. 4.1  Schematic representation of transport systems at the inner and outer blood-retinal barrier (BRB). The BRB consist of complex tight junctions of retinal capillary endothelial cells (inner BRB) and retinal pigment epithelial cells (outer BRB). The transport systems at the BRB can be classified into three categories; (1) blood-to-retina influx transport processes, (2) efflux pumps, and

(3) retina-to-blood efflux transport processes. In secondary active transport, the abluminal/apical transporters in the blood-to-retina influx transport process and the luminal/basolateral transporters in the retina-to-blood efflux transport process, which are indicated by a question mark, are not well characterized. GLUT facilitative glucose transporter; LAT L-type amino acid transporter; MCT H+- coupled monocarboxylate transporter; SMCT Na+-coupled monocarboxylate transporter; P-gp P-glycoprotein; MRP multidrug resistance-associated protein; BCRP breast cancer resistance protein; Oat organic anion transporter; Oatp organic anion transporting polypeptide

to develop the systemic route for efficient retinal drug delivery. In this chapter, we present a potential approach of the BRB-targeted retinal drug delivery through an overview of transport systems that are expressed at the BRB.

4.1.1  Role of the Blood-Retinal Barrier as a Dynamic Interface

The BRB consists of retinal vascular endothelial cells (RVEC: inner BRB) and retinal pigment epithelial (RPE) cells (outer BRB) (Fig. 4.1). The inner BRB is responsible for nourishment of the inner two-thirds of the retina whereas the outer BRB is responsible for nourishment of the remaining one-third of the retina

(Hosoya and Tomi 2005). Essential nutrients for neuronal cells, e.g., ganglion cells, bipolar cells, horizontal cells, amacrine cells, and Müller glial cells are supplied mostly across the inner BRB whereas those for photoreceptor cells are supplied across the outer BRB. RVEC and RPE cells form tight monolayers with complex tight junctions which prevent or decrease nonspecific diffusion across the monolayer. Both cell types are well polarized. The luminal plasma membrane of RVEC is in contact with blood whereas the abluminal membrane faces the retina. Similarly, the basolateral plasma membrane of the RPE cells is in contact with choroidal blood and the apical membrane faces the retina. Thus, the concerted actions of transporters which are localized in different membranes of RVEC and RPE cells enable the vectorial transport of a variety of compounds in the blood-to-retina and the retina-to-blood directions.

4.1.2  Potential Approach of Blood-Retinal Barrier-Targeted Systemic Drug Delivery to the Retina

A number of parameters need to be considered for systemic drug delivery to the retina: retinal blood flow, influx and efflux transport systems at the BRB, protein binding in the blood, clearance from the blood, and activity of drug metabolizing enzymes in peripheral tissues, blood, and at the BRB. Recent progress in the BRB research has revealed that multiple transporters/receptors are expressed at the BRB. This has opened the door to the development of the BRB-targeted drug delivery to the retina because drug recognition by the BRB transporters/receptors would greatly influence the disposition into the retina. Figure 4.1 illustrates three kinds of transport systems at the BRB. One group represents the blood-to-retina influx transport systems that supply nutrients such as glucose, amino acids, nucleosides, monocarboxylates, and vitamins to retinal cells. Some transporters transport not only their physiologic substrates but also therapeutic drugs that bear structural resemblance to their physiological substrates. Designing amino acidmimetic drugs which are recognized by amino acid transporters at the BRB is a promising approach to achieve retinal drug delivery. Thus, the influx transport systems at the BRB may have potential as a drug delivery route for the treatment of retinal diseases. The second group consists of the efflux pumps that prevent entry of xenobiotics into the RVEC and RPE cells by pumping them out back into the circulating blood. These efflux systems are located in the luminal and basolateral membranes of RVEC and RPE cells, respectively. Especially for hydrophobic drugs that penetrate the barrier mostly by passive diffusion, we need to consider that these efflux processes may contribute to the restricted distribution of drugs to the retina. The third group represents the retina-to-blood efflux transport systems that act to eliminate metabolites and neurotoxic compounds from the retina. To evaluate the ability of various pharmacologic agents to penetrate the BRB, it would be necessary to consider the combined net result of two different

processes,­ namely, the uptake of these agents into the RVEC or RPE cells via influx transporters and their subsequent excretion into the blood via efflux transporters. For example, the retina-to-blood efflux transport of several organic anions has been supposed to involve the concerted actions of organic anion transporter 3 (OAT3, SLC22A8) and multidrug resistance-associated protein 4 (MRP4, ABCC4) at the inner BRB (Barza et al. 1983; Hosoya et al. 2009). OAT3 and MRP4 share substrates­ such as b-lactam antibiotics and the anticancer drug 6-mercaptopurine (6-MP). In this case, inhibition of drug efflux transporters may lead to an increased distribution of drug to the retina. Studies carried out by Kompella and his coworkers­ have demonstrated that preadministration­ of probenecid, an organic anion transporter inhibitor, increases retinal concentration of N-4-benzoylam­ inophenylsulfonylglycine (BAPSG), a novel anionic aldose reductase inhibitor (Sunkara et al. 2010). Taken collectively, development of drugs that are well distributed to the retina can be achieved by incorporating structures that are recognized by the blood-to-retina transport systems or are not recognized by the retina-to-blood efflux systems. The success of retinal drug delivery may thus depend on several factors: (1) identity of the transporters that are expressed specifically at the BRB, (2) differential localization of the transporters in the two poles of the plasma membrane of the RVEC and RPE cells, and (3) substrate selectivity of the individual transporters, particularly differences in substrate specificity between the influx transporters and the efflux transporters. These factors can be exploited to our advantage to establish efficient strategies for optimal delivery of clinically relevant therapeutic drugs into the retina (Mannermaa et al. 2006; Hosoya and Tachikawa 2009).

4.2Blood-Retinal Barrier Influx Transporters/Receptors as a Potential Route for Retinal Drug Delivery

The BRB transporters play an essential role in the blood-to-retina transport of essential nutrients such as glucose, amino acids, vitamins, and nucleosides. The role of transporters in this process has been assessed by the greater blood-to-retina permeability rates of these essential nutrients compared with that of mannitol, a marker of passive non-carrier-mediated diffusion (Hosoya and Tachikawa 2009). The molecular identity of the transporters at the BRB has been established using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB cells) as in vitro model of inner BRB (Hosoya et al. 2001b) and primary cultures and cell lines of RPE cells as in vitro model of outer BRB. A considerable amount of work on the transport characteristics of RPE cells has also been carried out using the ARPE-19 cell line. Apical membrane vesicles from RPE cells and isolated RPE/choroid preparations have also been used for the directional transport studies. With the use of these various approaches, a great deal of information is now available on the identity and characteristics of transporters at the BRB as summarized in Table 4.1.

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