Influx Transport of Cationic Drug at the Blood–Retinal Barrier: Impact on the Retinal Delivery of Neuroprotectants

Abstract

The retina is a tissue essential for vision, and the blood–retina barrier (BRB) helps to maintain an optimal microenvironment for the neural system in the retina. Recent findings concerning the BRB showed the involvement of transporters at the inner and outer BRB in drug and nutrient transport, suggesting their utility in the development of novel drug delivery systems to the retina. An in vitro–in vivo relationship study of permeability suggested the influx transport of verapamil, a cationic drug, across the BRB, and further in vivo and in vitro studies of cationic drugs, such as verapamil, propranolol and clonidine, revealed the involvement of carrier-mediated process in their influx transport at the BRB. Studies on substrate specificity in TR-iBRB2 cells, an in vitro model cell line of the inner BRB, suggests the involvement of novel organic cation transporter in the influx transport of cationic drugs at the inner BRB. Considering the neuroprotective effect previously reported for several cationic drugs, such as propranolol and clonidine, the study of cation transport at the BRB is widely expected to improve the treatment of retinal diseases, such as diabetic retinopathy and age-related macular degeneration.

1. INTRODUCTION

The retina is an essential neural tissue involved in vision: visual information is processed by the retinal–neural system formed by the neural cells, such as rod cells, cone cells, bipolar cells, ganglion cells, horizontal cells and amacrine cells, and by Müller cells that are the retinal glial cells (Fig. 1). For the normal function of the retinal–neural system, the retinal microenvironment is required to be optimally sustained, suggesting the physiological importance of the blood–retinal barrier (BRB) in regulating material transport between the retina and circulating blood.^1,2) In the retina, retinal capillary endothelial cells and retinal pigment epithelial (RPE) cells are responsible for the inner and outer BRB, respectively. The tight junction formed by these cells strictly limits non-specific material transport through the paracellular route, suggesting that transcellular transport significantly contributes to the supply of essential nutrients and the elimination of endobiotics and xenobiotics.^2,3)

Fig. 1. Structure of the Blood–Retinal Barrier (BRB)

The retinal capillary endothelial cells (inner BRB) and the retinal pigment epithelial cells (RPE cells, outer BRB) are responsible for the structure and function of the BRB. The tight junction (TJ) formed by these cells limits the paracellular material transport at the BRB, suggesting the physiological and pharmacological significance of influx and efflux transport systems at the BRB.

In recent years, remarkable progress has been made in the study of transport at the BRB. The transcellular transport of various compounds at the BRB has been shown to involve carrier-mediated transport process, suggesting the major contribution of transporter molecules in the retinal capillary endothelial cells and RPE cells. As typical examples, studies of the inner BRB have suggested the role of transporters, such as glucose transporter (GLUT1/solute carrier 2A1 (SLC2A1)), L-type amino acid transporter (LAT1/SLC7A5), cationic amino acid transporter 1 (CAT1/SLC7A1), Na⁺-dependent multivitamin transporter (SMVT/SLC5A6), equilibrative nucleoside transporter 2 (ENT2/SLC29A2) and taurine transporter (TauT/SLC6A6), in supplying nutrients into the retina.^4–10) Other studies have shown the expression of ATP-biding cassette (ABC) transporters, such as P-glycoprotein (P-gp/MDR1/ABCB1), multidrug resistance associated protein 4 (MRP4/ABCC4) and breast cancer resistance protein (BCRP/ABCG2), and suggest their contribution to the elimination of metabolites and xenobiotics.^11–13)

These research achievements suggest that various transporters contribute to maintaining a suitable retinal microenvironment for the retinal–neural system. Investigation of facilitative material transport at the BRB is widely expected to improve the treatment of such retinal diseases as diabetic retinopathy and age-related macular degeneration, since carrier-mediated transport at the BRB appears to be highly promising in the development of efficient and safe drug delivery systems to the retina.

2. IN VITRO–IN VIVO RELATIONSHIP IN TRANSPORT ACROSS THE BRB

In the study of material transport across the BRB, the retinal uptake index (RUI) has been viewed as a useful in vivo method to evaluate the permeability of compounds at the BRB.¹⁴⁾ RUI is available from a calculation based on data experimentally obtained from carotid artery injection of compounds with a diffusible internal reference, such as [¹⁴C]n-butanol; RUI expresses the fractional uptake of the test compound in the retina as a percentage of the fractional uptake of the diffusible internal reference compound. However, RUI is considered to be an adverse method in the investigation of multiple samples; we need to establish a quick and easy in vitro method for the high throughput screening conducted in industrial drug discovery, clearly suggesting the need to clarify the in vitro–in vivo relationship of membrane permeability at the BRB.

To date, permeability studies involving RUI and in vitro cell uptake have shown that TR-iBRB2 cells, an in vitro model cell line of the inner BRB, are useful in evaluating the permeability of drugs and nutrients at the BRB.^13,15–17) The latest results show a correlation between the initial uptake rate (log V) obtained in TR-iBRB2 cells and RUI of compounds undergoing carrier-mediated transport and passive diffusion, respectively, and exhibited trend lines: Eq. 1 (RUI=26.5× exp(0.887× log V)) and Eq. 2 (RUI=26.5× exp(1.55× log V))¹⁷⁾ (Figs. 2A, B), suggesting the great utility of TR-iBRB2 cells in drug discovery for treating the retinal diseases.

Fig. 2. In Vitro–In Vivo Relationship Study in the Retina and Brain

The correlation of an initial uptake rate (log V) of compounds undergoing carrier-mediated transport (A) and passive diffusion (B) in TR-iBRB2 cells with the RUI in rats was studied, and a linear relationship described by Eqs. 1 and 2 are shown for compounds plotted in panels (A) and (B), respectively. The graphs of lipophilicity (log DC) and the tissue uptake index of compounds in the retina (C) and brain (D) of rats include lipophilicity trend lines described by Eqs. 3 and 4, which were separately constructed for the RUI and BUI of the compounds undergoing passive diffusion, respectively. Black symbols, gray squares and gray triangles represent cationic drugs, compounds undergoing carrier-mediated transport, and compounds undergoing passive diffusion, respectively. Diamonds represent substrates of P-gp, and white circles in panels (A) and (B) express compounds omitted in panels (C) and (D) for the purpose of illustration. 1, D-Glucose; 2, L-arginine; 3, L-phenylalanine; 4, L-leucine; 5, L-dopa; 6, biotin; 7, creatine; 8, D-mannitol; 9, testosterone; 10, corticosterone; 11, digoxin; 12, vincristine. The figure was prepared by reference to previous reports.^{7,16,17,21,24,25)} PAH, p-aminohippuric acid.

In an early verification study of the in vitro–in vivo relationship, test compounds were classified into two groups: compounds undergoing passive diffusion (Group I), and carrier-mediated transport (Group II) (Table 1). Group I exhibited a lipophilicity trend line (Eq. 3; RUI=46.2× exp (0.515× log DC)) derived from both lipophilicity (log DC) and in vivo permeability (RUI)¹⁶⁾ (Fig. 2C). Similarly, in the study of Group I at the BBB, a lipophilicity trend line (Eq. 4; BUI=24.2× exp(0.816× log DC)) was also shown for the log DC and the brain uptake index (BUI) that expresses the fractional uptake of the test compound in the brain, as with the RUI¹⁶⁾ (Fig. 2D). Regarding Group II, the measured RUI values of D-glucose, L-arginine, L-leucine, L-phenylalanine and biotin were shown to be greater than the values predicted by means of Eq. 3, and similar results were also observed for the BUI¹⁶⁾ (Figs. 2C, D), suggesting the involvement of transporters, such as GLUT1, CAT1, LAT1 and SMVT, expressed at the BRB and BBB in the influx transport of nutrients into the retina and brain (Fig. 3A). In addition, the involvement of LAT1 was suggested in the influx transport of L-dopa for the treatment of Parkinson’s disease across both the BRB and BBB, since L-dopa is a derivative of amino acid and its measured RUI and BUI was greater than predicted¹⁶⁾ (Figs. 2C, D).

Table 1. Classification of Compounds in the in Vitro–in Vivo Relationship Study

Classification	Compounds
Group I	Diazepam, Progesterone, Testosterone, Corticosterone, Warfarin, Antipyrine, Valproic acid, Dopamine, Uracil, Creatinine, D-Mannitol, PAH, MeAIB
Group II	T4, Adenosine, Acethyl-L-carnitine, L-Dopa, L-Phenylalanine, L-Leucine, Biotin, L-Arginine, D-Glucose, Glycine, Vincristine, Digoxin, Verapamil

This table was prepared by reference to previous reports.^16,17)

Fig. 3. Transport of Nutrient and Cationic Drug across the BRB

The combination of in vivo and in vitro analyses has shown the involvement of transporters in supplying nutrients, such as amino acids, sugar and vitamins, to the retina (A). In addition, recent studies suggest the involvement of facilitative transport system in the influx transport of cationic drugs at the BRB, although the expression of P-gp at the BRB is known (B).

Verapamil is a cationic drug, and its measured RUI value was also much greater than predicted, although its measured BUI was comparable to that predicted,¹⁶⁾ suggesting a marked difference of barrier function between the BRB and BBB. Verapamil, vincristine and digoxin are known as the typical drug substrates of P-gp, of which expression at the BRB was previously reported^12,13); their function at the BRB is clearly supported by the results in which vincristine and digoxin exhibited lower RUI values than those predicted¹⁶⁾ (Fig. 2C). These results show that the BRB and BBB function differently for verapamil, suggesting the facilitative influx transport of verapamil at the BRB (Fig. 3B).

3. CATIONIC DRUG TRANSPORT AT THE BRB

Several cationic drugs, such as memantine, propranolol and clonidine, have recently been reported to exert a neuroprotective effect in the brain and retina,^18–20) and these reports support the substantial contribution of the study of cationic drug transport at the BRB in developing future treatments for retinal disease. In the in vivo transport study of verapamil in rats, the apparent influx clearance of [³H]verapamil (K_{in, verapamil, retina}) was calculated to be 614 µL/(min·g retina), which was much greater than that of paracellular transport markers,²¹⁾ supporting the facilitative influx transport system of verapamil at the BRB. In the in vivo inhibition study of [³H]verapamil transport, the RUI was significantly reduced in the presence of pyrilamine, a cationic drug, whereas the BUI was increased by pyrilamine.²¹⁾ In the further assessment of differences in the BRB and BBB, a study with P-gp knockout rats showed the negligible impact of P-gp on K_{in, verapamil, retina} in spite of a marked alteration of the K_{in, verapamil, brain}²²⁾; similar results were reported in a study using P-gp/Bcrp knockout mice.²³⁾ These findings strongly support differences in transport properties of the BRB and BBB, and suggest the dominant influx transport of verapamil at the BRB (Fig. 3B).

Because of the neuroprotective effects of cationic drugs, as previously reported,^18–20) the influx transport system of cationic drugs at the BRB is expected to be helpful in the delivery of neuroprotectants into the retina. Among such cationic neuroprotectants, propranolol reduces the expression of vascular endothelial growth factor (VEGF), and clonidine induces basic fibroblast growth factor (bFGF) to exert a neuroprotective effect.^19,20) In the in vivo transport study, the measured RUI values of [³H]propranolol and [³H]clonidine were much greater than predicted values (Fig. 2C), and were inhibited in the presence of cationic drugs, such as verapamil and pyrilamine.^24,25) These results suggest the involvement of facilitative transport system in the influx transport of propranolol and clonidine at the BRB, supporting the utility of these transport systems in the efficient and safe delivery of cationic neuroprotectants to the retina.

4. CARRIER-MEDIATED TRANSPORT OF CATIONIC DRUGS AT THE INNER BRB

Referring to the influx transport of cationic drugs including neuroprotectants at the BRB, detailed investigations will likely contribute to our understanding of drug distribution and drug–drug interaction in the retina. In the study with TR-iBRB2 cells, the uptake of [³H]verapamil occurred in a time-, temperature- and concentration-dependent manner²¹⁾ (Fig. 4A), and these results suggest the involvement of carrier-mediated process in the influx transport of verapamil at the inner BRB. Similarly, in the in vitro uptake study of [³H]propranolol and [³H]clonidine, these drugs also revealed time-, temperature- and concentration-dependent transport properties, suggesting the involvement of carrier-mediated process in the influx transport of cationic neuroprotectants into the retina. Figure 4B shows the results obtained in the concentration-dependent uptake of [³H]clonidine by TR-iBRB2 cells.^24,25) In addition, the uptake study of pyrilamine by TR-iBRB2 cells also suggests the involvement of carrier-mediated processes, including high- and low-affinity transport, in the influx transport of pyrilamine across the inner BRB.²¹⁾ Table 2 presents the kinetic parameters suggested for cationic drug transport at the inner BRB.

Fig. 4. In Vitro Study of Cationic Drug Transport at the Inner BRB

The uptake study of [³H]verapamil (A) and [³H]clonidine (B) was performed by means of TR-iBRB2 cells, an in vitro model cell line of the inner BRB, and the concentration-dependent transports suggest the involvement of carrier-mediated transport process in the influx transport of verapamil and clonidine across the inner BRB. (C) The substrate specificity of [³H]verapamil transport was examined in TR-iBRB2 cells, and the results suggest the involvement of novel organic cation transporters in the influx transport of cationic drug across the inner BRB. Each point and bar represents the mean ± S.E.M. (n=3). * p<0.01, significantly different from the control. The figure was prepared by reference to Kubo Y, Kusagawa Y, Tachikawa M, Akanuma S, Hosoya K, “Involvement of a novel organic cation transporter in verapamil transport across the inner blood–retinal barrier,” 30, 847–856 (2013)²¹⁾ with permission from Springer; and to Kubo Y, Tsuchiyama A, Shimizu Y, Akanuma S, Hosoya K. “Involvement of carrier-mediated transport in the retinal uptake of clonidine at the inner blood–retinal barrier,” 11, 3747–3753, (2014)²⁵⁾ with permission from ACS Publications. PAH, p-aminohippuric acid.

Table 2. Kinetic Parameters Suggested for Cationic Drug Transports at the Inner BRB

Drugs		Km (µM)	Vmax (nmol/(min·mg protein))	Vmax/Km (µL/(min·mg protein))
Verapamil		61.9	3.31	53.5
Pyrilamine	High affinity	20.2	0.837	41.4
	Low affinity	252	22.3	88.5
Propranolol		237	11.9	50.2
Clonidine		287	43.7	152

This table was prepared by reference to previous reports where kinetic parameters were estimated in the in vitro uptake study in TR-iBRB2 cells.^21,24,25)

In the in vitro inhibition study, the substrate specificity of cationic drug transport at the inner BRB was investigated. In TR-iBRB2 cells, the uptake of [³H]verapamil was significantly inhibited by cationic drugs, including quinidine, pyrilamine, mecamylamine, amantadine, propranolol and clonidine, whereas no significant effect was shown by p-aminohippuric acid (PAH), a typical anionic compound (Fig. 4C), suggesting the involvement of organic cation transporter in verapamil transport at the inner BRB.²¹⁾ Similar results were obtained for [³H]propranolol and [³H]clonidine, suggesting that the transports of propranolol and clonidine also involve organic cation transporter at the inner BRB. Furthermore, the uptakes of [³H]verapamil, [³H]propranolol, [³H]clonidine and [³H]pyrilamine were significantly inhibited by cationic neuroprotectants, such as desipramine and memantine,^21,24,25) strongly suggesting that the delivery of neuroprotectants through carrier-mediated cationic drug transport is a promising way to improve the treatment of retinal diseases.

Based on functional properties, including substrate specificity and ion-sensitivity, the cationic drug transport at the BRB is assumed to involve a number of organic cation transporters (Fig. 5). The in vitro inhibition study revealed that substrate specificity of the cationic drug transport at the BRB is different from those of well-characterized organic cation transporters, such as plasma membrane monoamine transporter (PMAT/SLC29A4/ENT4), multidrug and toxin extrusion (SLC47/A1), organic cation/carnitine transporters (OCTNs/SLC22A4–5) and organic cation transporters (SLC22A1–3),^21,24,25) suggesting the involvement of novel organic cation transporters in the influx transport of cationic drugs across the BRB.

Fig. 5. Putative Transporters Involved in Cationic Drug Transport at the BRB

The in vitro study of substrate specificity in TR-iBRB2 cells suggests the involvement of novel organic cation transporters in the carrier-mediated influx transport of cationic drugs at the inner BRB. The study of ion-dependence suggested the involvement of an H⁺/organic cation antiporter in the influx transport of pyrilamine and propranolol.

5. CONCLUSION

In 1913, Schnaudigel proposed the barrier structure between the circulating blood and retina, and a century-old study has been demonstrated for the BRB.²⁶⁾ From the 1960 s onward, the BRB was thought to be a static barrier to limit non-specific material transport between the blood and retina, based on the tight junction formed by the responsible cells, such as capillary endothelial cells and RPE cells. However, because the retina is a small neural tissue, it has been difficult to assess in detail material transport in the retina. From the 2000 s onward, TR-iBRB2 cells, an in vitro model cell line, were established as useful in in vitro transport studies involving the BRB.^13,15) Transport and expression analyses by means of in vivo, in vitro and molecular biological methods have permitted us to challenge the details of drug and nutrient transport at the BRB. This review has presented the utility of TR-iBRB2 cells in drug discovery, in distinguishing different transport functions between the BRB and BBB, and in elucidating cationic drug influx transport at the BRB. Although recent findings have implied the possible involvement of lysosomal trapping (the sequestration of drugs in lysosomes) in drug permeability at the BRB, the uptake study of lysosomal trapping probe suggested that lysosomal trapping is negligible in the transport of cationic drugs, including neuroprotectants, at the inner BRB.²⁷⁾ Therefore, further detailed study of the function and expression of transporters at the BRB is expected to make a large contribution toward improving the treatment of retinal diseases.

Acknowledgments

This review was based upon work supported in part by Grants-in-Aid from the Japan Society for the Promotion of Science (JSPS KAKENHI Grant Number 17K08409 and 16H05110), the Uehara Memorial Foundation, the Tamura Science and Technology Foundation, the Takeda Science Foundation, the Suzuki Memorial Foundation, the Nakatomi Foundation, and the Mochida Memorial Foundation for Medical and Pharmaceutical Research.

Conflict of Interest

The authors declare no conflict of interest.

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