ABC ATP-binding cassette; AP apical membrane; b bovine; BL basolateral membrane; BRB bloodretinal barrier; h human; LU luminal membrane; m mouse; rt rat; SLC solute carrier

4.2.1  Amino Acid-Mimetic Drugs

L-DOPA [levodopa, (-)-3-(3,4-dihydroxyphenyl)-L-alanine], the amino acid precursor of dopamine, and amino acid mustards are examples of amino acid mimetic drugs. The Na+-independent amino acid transport system, system L recognizes neutral amino acids as endogenous substrates. At the molecular level, system L is encoded by L (Leucine-referring)-type amino acid transporter (LAT) 1 (SLC7A5) and LAT2 (SLC7A8). Retinal capillary endothelial cells exclusively express LAT1

protein (Tomi et al. 2005). RPE cells express mRNAs for LAT1 (Uchino et al. 2002) and LAT2 (Nakauchi et al. 2003). LAT1 mRNA expression in ARPE-19 cells is quantitatively 42.5-fold higher than that of LAT2 mRNA expression (Yamamoto et al. 2010). Indeed, functional analysis suggests that the contribution of LAT1 to L-leucine uptake by ARPE-19 cells is 70% of the total L-leucine uptake (Yamamoto et al. 2010). Many patients with Parkinson’s disease have blurred vision or other visual disturbances, which are reflected in the reduced retinal dopamine concentration and delayed visual evoked potentials (Bodis-Wollner 1997). L-DOPA corrects these deficiencies (Bhaskar et al. 1986). Since LAT1 transports L-DOPA as a high-affinity substrate with the Michaelis constant of 34.2 mM (Uchino et al. 2002), LAT1 at the BRB provides an important route for the delivery of L-DOPA into the retina. Melphalan (phenylalanine mustard), an alkylaing agent used in the treatment of retinoblastoma (Kaneko and Suzuki 2003), and gabapentin (an analog of g-aminobutyrate), a drug used in the treatment of acquired pendular nystagmus (Averbuch-Heller et al. 1997), are also transported via LAT1 (Goldenberg et al. 1979). Tomi et al. (2005) and Hosoya et al. (2008a) have investigated the potential participation of LAT1 in the retinal delivery of various amino acid mustards as alkylating agents using TR-iBRB cells. Since LAT1 is an obligatory amino acid exchanger, influx of one amino acid substrate into cells is coupled obligatorily to efflux of some other amino acid substrate. Interestingly, melphalan did not trans- stimulate the efflux of [3H]phenylalanine and [3H]L-leucine in TR-iBRB cells (Hosoya et al. 2008a) and ARPE19 cells (Yamamoto et al. 2010), respectively. This suggests that melphalan may not be a good substrate for LAT1. In support of this notion, melphalan needs to be injected into the vitreous humor in patients with retinoblastoma because it is not efficiently transported from the blood to the retina through the BRB. In contrast, phenylglycine-mustard was very effective in inducing the efflux of [3H]phenylalanine, suggesting that phenylglycine-mustard is an effective transportable substrate for LAT1. Even though L-DOPA and certain amino acid-mustards are recognized as transportable substrates for LAT1, LAT1-mediated delivery of drugs into the retina is likely to exhibit competition from its endogenous amino acid substrates in vivo. LAT1 possesses high affinity for its endogenous substrates; the Michaelis constants for L-leucine in TR-iBRB cells and ARPE19 cells are ~15 mM (Tomi et al. 2005) and 8.7 mM (Yamamoto et al. 2010), respectively. The normal plasma concentration of L-leucine (80–160 mM) is several-fold higher than this value. Furthermore, the other endogenous amino acids such as L-isoleucine, L-valine, L-phenylalanine, L-tyrosine, and L-tryptophan, which are transportable substrates for LAT1, are present in the plasma at the concentration range of 52–220 mM. These amino acids in plasma may saturate LAT1-mediated transport at the BRB. RPE cells express mRNA for y+LAT1 (SLC7A7) (Nakauchi et al. 2003), which transports cationic amino acids such as L-arginine and L-lysine in an Na+- independent manner and neutral amino acids in an Na+-dependent manner. Under physiologic conditions, the transport process mediated by y+LAT1 involves Na+- dependent entry of neutral amino acids into cells coupled with exit of cationic amino acids from the cells. Therefore, there may be a functional coupling between LAT1/ LAT2 and y+LAT1 in the vectorial transfer of amino acid-mimetic drugs across the

outer BRB, but the localization of these transporters needs to be investigated. RPE cells also express the Na+- and Cl−-dependent amino acid transporter ATB0,+ (SLC6A14) (Nakanishi et al. 2001). ATB0,+ transports a wide variety of drugs and prodrugs (Ganapathy and Ganapathy 2005), including nitric oxide synthase inhibitors (Hatanaka et al. 2001) and amino acid derivatives of antiviral agents such as valacyclovir (Hatanaka et al. 2004) and valganciclovir (Umapathy et al. 2004).

4.2.2  Monocarboxylic Drugs

Endogenous monocarboxylates such as lactate, pyruvate, and ketone bodies (b- hydroxybutyrate and acetoacetate) are transported via two types of transporters, e.g., the H+-coupled monocarboxylate transporters (MCTs) belonging to the SLC16 family and the Na+-coupled monocarboxylate transporters (SMCTs) belonging to the SLC5 family. MCT1 (SLC16A1) is localized in both the luminal and abluminal membranes of RVEC (Gerhart et al. 1999). RPE cells express MCT1, MCT3 (SLC16A8), and SMCT1 (SLC5A8). MCT1 is expressed exclusively in the apical membrane of RPE cells whereas MCT3 is expressed exclusively in the basolateral membrane (Philp et al. 1998, 2003; Daniele et al. 2008; Deora et al. 2005). SMCT1 is expressed only in the basolateral membrane of RPE cells (Martin et al. 2007). Thus, MCT1 at the inner BRB and SMCT1 at the outer BRB can be exploited to take up their substrates from the circulating blood into the cells. Several monocarboxylic drugs have been shown to be substrates for MCT1; this includes foscarnet, salicylate, benzoate, and a prodrug of gabapentin (Enerson and Drewes 2003; Morris and Felmlee 2008). The monocarboxylic drugs that are recognized by SMCT1 include benzoate, salicylate, 5-aminosalicylate (Gopal et al. 2007), and 3-bromopyruvate (Thangaraju et al. 2009). Therefore, MCT1 and SMCT1 at the BRB have potential for the delivery of monocarboxylic drugs into retina. Nonsteroidal anti-inflamma- tory drugs (e.g., ibuprofen, ketoprofen), which are also monocarboxylates, are not recognized by SMCT1 as transportable substrates, but these drugs function as blockers of the transporter (Itagaki et al. 2006). A study on MCT3 knockout mice reveals that disruption of MCT3 gene impairs visual function presumably as a consequence of reduction in subretinal space pH (Daniele et al. 2008). This suggests that MCT3 at the outer BRB is critically positioned to facilitate transport of lactate out of the retina and plays a role in pH homeostasis of the retina (Daniele et al. 2008).

4.2.3  Nucleoside Analogs

The Na+-independent equilibrative nucleoside transporters (ENTs) belonging to the SLC29 family accepts purine and pyrimidine nucleosides as endogenous substrates. ENT1 (SLC29A1) and ENT2 (SLC29A2) transport several antiviral and anticancer nucleoside drugs such as 2¢, 3¢-dideoxycytidine (zalcitabine, ddC),

2¢,3¢-dideoxyinosine (ddI), cytarabine, and gemcitabine (Baldwin et al. 2004; Yao et al. 2001). ENT2 also transports 3¢-azido-3¢-deoxythymidine (zidovudine, AZT) (Baldwin et al. 2004; Yao et al. 2001). The blood-to-retina transport of [3H]adenosine, a purine nucleoside, is carrier-mediated and is inhibited competitively by unlabeled adenosine and thymidine but not by cytidine (Nagase et al. 2006). Similar features are evident for adenosine transport in TR-iBRB cells which express ENT2 mRNA (Nagase et al. 2006). Adenosine plays an important role in retinal neurotransmission, blood flow, vascular development, and cellular response to ischemia. The delivery of adenosine into the retina across the inner BRB is therefore an important physiological process. The Michaelis constant for adenosine for transport via ENT2 is ~30 mM, which is much higher than adenosine concentration in plasma (~0.1 mM), indicating that ENT2 is not saturated with adenosine under physiologic conditions. Since the nucleoside analogs described earlier are substrates for ENT2 (Baldwin et al. 2004; Yao et al. 2001), this transporter is potentially involved in the delivery of such drugs into the retina. Although the expression of ENTs has not been examined in RPE cells, ARPE-19 cells exhibit nitrobenzylmercaptopurine riboside-sensitive uptake of nucleoside (Majumdar et al. 2004). Therefore, it is very likely that the nitrobenzylmercaptopurine-sensitive nucleoside transporter ENT1 is expressed in these cells. ENTs at the BRB may be a potential route for the delivery of nucleoside drugs from the circulating blood to the retina.

4.2.4  Folate Analogs

The reduced folate carrier (RFC1, SLC19A1), folate receptor a, and the H+- coupled folate transporter (PCFT, SLC46A1) play a role in the uptake of folate and its analogs (Zhao et al. 2009). Tetrahydrofolate functions as a cofactor for de novo synthesis of purines and pyrimidines, and also as a critical component in the metabolism of the sulfur-containing amino acids methionine and homocysteine. Folate deficiency causes visual dysfunction; therefore, neural retina must possess mechanisms to obtain this essential vitamin. This implies that the cells constituting the BRB must express transport systems for folate. Since folate exists predominantly as the methyl derivative of the reduced folate (N5-methyltetrahydrofolate, MTF) in the plasma, MTF would be the principal substrate for the folate transport proteins. RFC1 mediates MTF uptake by TR-iBRB cells, being inhibited by folate analogs such as methotrexate and formyltetrahydrofolate (Hosoya et al. 2008b). RFC1 mRNA is expressed abundantly in freshly isolated rat RVEC (Hosoya et al. 2008b). The outer BRB is capable of transcellular transfer of folate in the blood-to-retina direction, indicating that the folate transport proteins are expressed in RPE cells in a polarized manner (Bridges et al. 2002). Folate receptor a is expressed in the basolateral membrane whereas RFC1 is expressed exclusively in the apical membrane (Chancy et al. 2000). Folate receptor a would be involved in the uptake of folate across the basolateral membrane as the first step in the folate transport across RPE cells from the circulating blood to the retina. RFC1 in the apical membrane will then