6.6  Transfer Through the Vitreoretinal Border

6.6.1  The Role of the Blood–Retinal Barrier

Passive penetration of substances into the retina is restricted by the blood retinal barrier (BRB) and this barrier can be divided into the inner and outer BRB. The inner BRB, positioned at the inner section of the retina, is closest to the vitreous and is formed by capillary endothelial cells, connected via tight junctions. The outer BRB consists of the melanin-rich, RPE. Blood is supplied to the initial two-thirds of the retina, closest to the vitreous, via the inner BRB and the rest is nourished by the choriocapillaris via the outer BRB.

Capillary endothelial cells surrounding the vessel lie within a network of neurons, astrocytes and Műller cells and control movement between the retina and blood supply (Gardner et al. 2000). This movement is controlled mainly via influx and efflux transporters, which ensure the retina receives a rich energy, supply of glucose, lactate and creatine, alongside antioxidants including vitamin C and cysteine and amino acids; leucine and taurine. Many of these transporters have low affinity and therefore respond to increased gradients by increasing flux – for example, lactate transport by monocarboxylate transporters (Hertz and Dienel 2004). Influx and efflux transporters are also responsible for controlling the movement of organic anions in and out of the retina (Hosoya et al. 2009a, b). Urtti and colleagues completed an extensive review of the role of transporters in the eye including those involved into the anterior tissues (Mannermaa et al. 2006). In view of this, we have specifically directed our discussion with regard to those transporters involved in movement of compounds from the vitreous into the retina.

6.6.1.1  Amino Acid Transport

The mechanisms of amino acid transport from the vitreous to the retina could have potential application in drug delivery and are therefore of relevance, although most observations are limited to cell culture models such as those described by Hosoyo et al. (2001). In the intact eye, microdialysis has been useful in elucidating the role of the large neutral amino acid transporter system (LAT) on the retinal uptake of L-phenylalanine (L-Phe). L-Phe was administered to rabbit eyes intravitreally, alone and also in the presence of known LAT inhibitors. Retinal uptake of L-Phe was shown to be inhibited following administration of the blockers, demonstrating the role of LAT in amino acid movement from the vitreous to the retina (Atluri et al. 2008). In addition, amino acid transporter system A is important in the vitreous to retina movement of proline. The rate of vitreous elimination of proline was shown to differ from that of the bulk flow marker and was indicative of active transport in the rat (Yoneyama et al. 2010).

6.6.1.2  P-Glycoprotein

The multidrug transporter P-glycoprotein (P-gp) is a member of the ATP-binding cassette (ABC) family of transporters and is involved in various functions including cell signalling, ion transport, nutrient uptake and efflux of waste compounds. A number of isoforms of P-gp have been identified including multidrug resistant 1 (MDR1) and multidrug resistant 2 (MDR2), shown to be expressed in human cell lines (Hennessy and Spiers 2007).

Earlier ocular studies examined the role of the MDR pump on the uptake of fluorescein sodium (FS) and benzcylamine phenyl sulfonylglcine (BAPSG) into human RPE cells. BAPSG is a selective aldosterone reductase inhibitor, considered for its potential role in the treatment of diabetic retinopathy. Indomethacin, verapamil and probenecid were co-administered with FS and BAPSG as known MDR inhibitors. Both indomethacin and probenecid were shown to increase FS accumulation in cells, with no significant effect demonstrated by verapamil. BAPSG accumulation also increased in the presence of indomethacin and probenecid but also verapamil when the concentration of inhibitor was increased to 10 mm. The efflux of BAPSG from RPE cells was also shown to be significantly higher when the inhibitors were not present (Aukunuru et al. 2001).

Using various RPE cell lines the penetration of rhodamine 123, a known P-gp substrate, into cells has been investigated. In one cell line, the presence of the P-gp inhibitor, verapamil, resulted in an increased uptake of rhodamine 123 by approximately 13-fold. Little effect was noted on the other cell lines studies, reportedly due to poor expression of P-gp (Constable et al. 2006). The appreciation of the importance of P-gp has prompted investigations of the inner blood–retinal-barrier cell line (TR-iBRB) transfected with P-gp. Uptake studies of rhodamine 123 were performed in the presence and absence of a series of test compounds; AGN 194716, AGN 195127, AGN 197075, acebutolol, alprenolol, atenolol, brimonidine, carbamazepine epoxide (CBZ-E), metoprolol, nadolol and sotalol, to identify potential P-gp inhibitors. Using TR-iBRB cell lines, rhodamine 123 uptake identified only AGN 197075 as an inhibitor of P-gp-mediated efflux of rhodamine 123 from compounds studied (Shen et al. 2003).

Steuer et al. described both P-gp and multidrug resistant protein (MRP) expression in the outer BRB in the pig. Verapamil and rhodamine 123 were applied to both the choroid and retina independently and a higher cell permeability of both compounds into cells was demonstrated when the compounds were applied to the retinal side of the outer BRB. Increases in permeability were 3.5-fold for verapamil and 2.6-fold for rhodamine, leading to the conclusion that P-gp expression in the outer BRB must be greater at the choroidal side. The authors also investigated the penetration of FS from retina to choroid in the presence and absence of the MRP inhibitor, probenecid. FS permeability increased 11-fold following addition of the inhibitor (Steuer et al. 2005).

6.6.1.3  Organic Cationic Transporters

Organic cationic transporters (Oct) are members of the solute carrier transporter gene family and are involved in the transport of small organic cations and hydrophilic

compounds (Kusuhara and Sugiyama 2004). Known substrates of Oct include tetraethylammonium and monoamine neurotransmitters (Tsuji 2005). The Oct has been shown to be expressed in mouse RPE (Rajan et al. 2000) and its involvement in retinal drug transport demonstrated (Han et al. 2001). Using human RPE cell lines, RPE/Hu and ARPE-19, the uptake of verapamil into RPE was shown to be a saturable process, with an apparent Km equal to 7.2 mM. The rate of verapamil uptake decreased in the presence of metabolic inhibitors, when the temperature was reduced, and in the presence of some organic cations: including quinidine, pyrilamine, quinacrine and diphenhydramine. Cationic drugs – diltiazem, timolol and propranolol – commonly used in the treatment of glaucoma also inhibited uptake. However, no change in uptake rate was seen in the presence of other organic cations, including tetraethylammonium and cimetidine, revealing the expression of a new Oct subtype.

6.6.1.4  Organic Anion Transporters

Organic anion transporters (Oat) are also members of a family of solute carrier transporters and involved in energy-independent efflux transport (Brasnjevic et al. 2009). Using polymerase chain reaction (PCR), rOat3 (rat organic anion transporter) was found to be expressed in the retina and retinal endothelial cells of rats. In the intact eye, a series of radio-labelled drug candidates were administered via intravitreal injection and concentration changes measured by microdialysis. P-aminohippuric acid, benzylpenicillin and 6-mercaptopurine showed a biexponential elimination pattern from the vitreous. The elimination rate of all three was reduced in the presence of rOat3 inhibitor, probenecid, demonstrating the role of rOat3 in drug efflux (Hosoya et al. 2001).

Oat involvement in transport was also shown for [3H]-estradiol 17-beta glucuronide ([3H]E17bG). The study was performed in rats using microdialysis with co-administered [14C]D-mannitol as a marker of bulk flow movement. Removal of both compounds from the vitreous followed a biexponential pattern. In the initial phases of drug elimination from the vitreous, the elimination rate was similar for both compounds. However, the second phase of decline differed, with elimination of [3H]E17bG shown to be significantly greater than that of [14C]D-mannitol, with elimination constants of 9.0 × 10−3/min for [3H]E17bG and 5.0 × 10−3/min for [14C]D-mannitol. The author suggested that the first phase represents the drug diffusing across the whole of the vitreous whereas the second phase, the true rate of drug elimination out of the vitreous. In the presence of probenecid, the rate of elimination of [3H]E17bG was reduced similar fluxes to that of [14C]D-mannitol, leading to the conclusion that [3H]E17bG undergoes efflux transport via a probenecid-sensitive organic anion transport process, likely to take place at the BRB (Hosoya et al. 2003).

6.6.1.5  Other Transporters

In the investigation of fluorescein and fluorescein monoglucuronide elimination from rabbit vitreous, an elimination rate for fluorescein was calculated to be 0.22 ± 0.03/h