advances in ocular drug delivery, 2012: 1-31 isbn: 978...

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Advances in Ocular Drug Delivery, 2012: 1-31 ISBN: 978-81-308-0490-3 Editor: Ashim K. Mitra

1. Transport of drugs across the inner and

outer blood-retinal barriers: Relevance of

transporters in the retinal blood vessel

endothelium and the retinal pigment

epithelium

Masanori Tachikawa1, Ken-ichi Hosoya1, Sylvia B. Smith2, Pamela M. Martin3 and Vadivel Ganapathy3

1Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences University of Toyama, Toyama, Japan; 2Department of Cellular Biology and Anatomy, Medical College

of Georgia, Augusta, Georgia, U. S. A.; 3Department of Biochemistry and Molecular Biology Medical College of Georgia, Augusta, Georgia, U. S. A.

Introduction

There is an urgent need for the development of optimal and efficient drug

delivery systems for the treatment of retinal diseases because of the potential

loss of vision if these diseases go untreated. Delivery of therapeutic drugs to

the retina poses various hurdles. Topical application of drugs (eye drops) is

ineffective in achieving therapeutically relevant concentrations of the drugs

in the retina due to the long diffusional distance and counter-directional

intraocular convection from the ciliary body to Schlemm’s canal [1].

Intravitreal delivery with implants and direct injections carries a high risk Correspondence/Reprint request: Dr. Masanori Tachikaw, Department of Pharmaceutics, Graduate School of

Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan

Masanori Tachikawa et al. 2

of deleterious side effects such as post-operative endophthalmitis,

hemorrhage, and retinal detachment. It is widely believed that systemic

administration is not an effective means to deliver drugs into the retina

because of the blood-retinal barriers. The retina is considered as a privileged

tissue protected by the inner and outer blood-retinal barriers from potentially

harmful chemicals and agents that may be present in the systemic circulation.

While this particular role of blood-retinal barriers is certainly beneficial to the

retina, it also poses a significant problem to deliver therapeutic drugs to the

retina via systemic administration. However, the blood-retinal barriers are not

impermeable structures; essential nutrients to support the growth and viability

of various cell types within the retina are transferred efficiently from

systemic circulation via these barriers. Recent progress in blood-retinal

barrier research has revealed that the specific cell types comprising these

barriers express a wide variety of transporters essential for the blood-to-

retinal influx of these nutrients. There are some transporters however that

contribute to the protective function of the blood-retinal barriers by mediating

the retina-to-blood efflux of toxins and drugs, thus playing an active role in

the removal of potentially harmful chemicals from the retina. It has become

increasingly clear in recent years that even though the physiologic function of

most of the transporters in mammalian cells is to facilitate transmembrane

movement of biologically relevant compounds such as amino acids,

monosaccharides, nucleosides, monocarboxylates, and vitamins, these

transporters do recognize several xenobiotics and therapeutic agents that

might bear structural resemblance to their physiological substrates. This

feature can be exploited to our advantage to develop efficient strategies for

optimal delivery of clinically relevant therapeutic drugs into the retina [2,3].

The blood-retinal barriers consist of retinal capillary endothelial cells (inner

blood-retinal barrier) and retinal pigment epithelial (RPE) cells (outer blood-

retinal barrier) (Fig. 1). Both these cell types form tight monolayers with

complex tight junctions between adjacent cells within the monolayer. This

prevents or decreases non-specific diffusion of chemicals across the

monolayer. The inner blood-retinal barrier is responsible for nourishment of

the inner two-thirds of the retina while the outer blood-retinal barrier is

responsible for nourishment of the remaining one-third of the retina [4].

Thus, essential nutrients for photoreceptor cells are supplied through transfer

across RPE from choroidal circulation whereas those for other neuronal cells

(e.g., ganglion cells, bipolar cells, horizontal cells, amacrine cells) and Muller

cells come mostly through transfer across the retinal capillary endothelial

cells. Since these two monolayers have to perform vectorial transfer of

nutrients in the blood-to-retina direction and also eliminate metabolic waste

products in the retina-to-blood direction, the capillary endothelial cells of the

Drug delivery to retina 3

Figure 1. Schematic representation of the retina and the inner and outer blood-retinal

barriers. The blood-retinal barriers form complex tight junctions of retinal capillary

endothelial cells (inner blood-retinal barrier) and retinal pigment epithelial cells (outer

blood-retinal barrier). GC, ganglion cell; AC, amacrine cell; BC, bipolar cell; HC,

horizontal cell; MC, Muller cell; RC, rod photoreceptor cell; CC, cone photoreceptor

cell; RPE, retinal pigment epithelial cell.

inner blood-retinal barrier and the RPE cells of the outer blood-retinal barrier

are polarized. The plasma membrane of the endothelial cells consists of a

luminal membrane that is in contact with blood and an abluminal membrane

that faces the retina. Similarly, the plasma membrane of the RPE cells

consists of a basolateral membrane that is in contact with the Bruch’s

membrane apposed to choroidal blood and an apical membrane that faces the

retina. The feasibility and success of drug delivery to the retina using

transporters depend on several factors: (i) identity of the transporters that are

expressed specifically in the inner and outer blood-retinal barriers, (ii)

differential expression of the transporters in the two poles of the plasma

membrane of the polarized cells that constitute these barriers, and (iii)

substrate selectivity of the individual transporters, particularly differences in

substrate specificity between the influx transporters and the efflux

transporters.


Influx transporters at the inner blood-retinal barrier

The retinal capillary endothelial cells that constitute the inner blood-

retinal barrier supply essential nutrients such as glucose, amino acids,

vitamins, and nucleosides to the retina. The role of transporters in this

process has been assessed by comparing the blood-to-retina permeability

rates of these essential nutrients with that of mannitol, a marker of passive

non-carrier-mediated diffusion. The nutrients have several-fold higher

permeability rates than mannitol (Table 1), implying that specific transporters

are involved in the blood-to-retina transfer of these compounds. The

molecular identity of the transporters responsible for the transfer of

individual nutrients has been established using a conditionally immortalized

rat retinal capillary endothelial cell line (TR-iBRB cells) [5]. The influx

transporters identified thus far in this cell line are listed in Fig. 2. Some of

these transporters transport not only their physiologic substrates but also

several xenobiotics and therapeutic drugs that structurally mimic the

endogenous substrates, suggesting that these transporters may have potential

as drug delivery systems for the treatment of retinal diseases.

Table 1. Comparison of the permeability rates of the blood-to-retina influx transport.

The influx permeability rate was determined by integration plot analysis after

intravenous injection of radiolabeled compound. †Influx permeability rate of

D-glucose (544 μL min-1 g retina-1) is calculated from the influx rate of D-

glucose (6.8 μmol min-1 g retina-1)/normal D-glucose concentration in rat

plasma (12.5 mmol/L [49]. * μL min-1 g eye-1.


Figure 2. Efflux and influx transporters expressed in retinal capillary endothelial cells

that constitute the inner blood-retinal barrier. DHA, dihydroascorbic acid; MTF, N5-

methyltetrahydrofolate.

Transporter-mediated drug delivery across the inner blood-

retinal barrier

Amino acid mimetic drugs

L-DOPA [levodopa, (-)-3-(3,4-dihydroxyphenyl)-L-alanine], the amino

acid precursor of dopamine, is a widely used drug for treatment of

Parkinson’s disease. Many patients with this disease have blurred vision or

other visual disturbances, which are reflected in the reduced retinal dopamine

concentration and delayed visual evoked potentials [6]. L-DOPA corrects

these deficiencies [7], indicating that this amino acid is transported into the

retina to serve as the precursor for dopamine synthesis to correct the

deficiency of dopaminergic neurotransmission associated with Parkinson’s

disease. L-DOPA is a neutral aromatic amino acid that has been shown to be

a substrate for the Na+-independent amino acid transporter LAT1 [L

(Leucine-Preferring) Amino acid Transporter 1; SLC7A5] [8]. This drug

crosses the blood-brain barrier efficiently and enhances dopaminergic

neurotransmission in the central nervous system in patients with Parkinson’s

disease. It is known that LAT1, which is expressed in the brain capillary

endothelial cells, is responsible for this transport process [9]. Since this

transporter is also expressed in retinal capillary endothelial cells [10], it

provides a potentially important route for the delivery of L-DOPA into the

retina. Melphalan (phenylalanine mustard), an alkylaing agent used in the

treatment of certain cancers, and gabapentin (an analog of -aminobutyrate),

a drug used in the treatment of variety of neuropathic pain, are also

transported via LAT1 [11]. Tomi et al [10] and Hosoya et al [12] have

investigated the potential participation of LAT1 in the retinal delivery of

various amino acid mustards as alkylating agents using TR-iBRB cell line as

a model for the inner blood-retinal barrier. Since LAT1 is an obligatory


amino acid exchanger, it is possible to determine directly whether or not a

compound serves as a transportable substrate for LAT1 by monitoring the

ability of the compound to induce the efflux of preloaded radiolabeled substrate

[3H]phenylalanine in these cells. Surprisingly, melphalan failed to induce the

efflux of [3H]phenylalanine in TR-iBRB cells, suggesting that this drug is not

an effective transportable substrate for LAT1. With the same criterion,

tyrosine-mustard, alanine-mustard, ornithine-mustard, and lysine-mustard were

also shown to be poorly transportable substrates. In contrast, phenylglycine-

mustard was very effective in inducing the efflux of [3H]phenylalanine,

suggesting that this particular amino acid mustard is an effective transportable

substrate for LAT1. Even though L-DOPA and certain amino acid-mustards are

recognized as transportable substrates for LAT1, other factors should be

considered to evaluate the efficacy of this transporter to deliver these drugs into

the retina in vivo. LAT1 possesses high affinity for its substrates; the Michaelis

constant for leucine in TR-iBRB cells is ~15 M [10]. The normal plasma

concentration of leucine is several-fold higher than this value (80-160 M).

Furthermore, LAT1 accepts not only leucine but also other branched chain

(isoleucine and valine) and aromatic (phenylalanine, tyrosine, and tryptophan)

amino acids as substrates, which are present in the plasma in considerable

quantities. Therefore, endogenous amino acid substrates are likely to decrease

the efficacy of LAT1-mediated delivery of drugs into the retina. This effect

may even be enhanced further under certain conditions such as high protein diet

that increases the plasma levels of branched chain and aromatic amino acids.

Nucleoside analogs

Several nucleoside analogs are currently used in the treatment of viral

infection and cancer. Examples of these analogs include 3’-azido-3’-

deoxythymidine (AZT), 2’, 3’-dideoxycytidine (ddC), 2’,3’-dideoxyinosine

(ddI), cladribine, cytarabine, fludarabine, gemcitabine, and capecitabine.

These drugs are transported into mammalian cells via nucleoside transporters,

which handle purine and pyrimidine nucleosides as their physiologic

substrates. 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 [13]. Similar features are

evident for adenosine transport in TR-iBRB cell line [13]. Molecular analysis

of nucleoside transporters expressed in this cell line revealed that the Na+-

independent equilibrative nucleoside transporter ENT2 (SLC29A2) is

expressed in these cells [13]. 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 blood-


retinal barrier is therefore an important physiological process. The Michaelis

constant for adenosine for transport via ENT2 is ~30 M, which is much higher

than adenosine concentration in plasma (~0.1 M), indicating that ENT2 is not

saturated with adenosine under physiologic conditions. Since the nucleoside

analogs mentioned above are substrates for ENT2 [14,15], the transporter

potentially plays a role in the delivery of such drugs into the retina.

Antioxidants

Retina is subjected to oxidative insult due to light exposure. Therefore,

this tissue has an obligate need for antioxidants for protection against light-

induced damage to the cells. In addition, many retinal diseases have oxidative

damage as an underlying pathological component. This includes diabetic

retinopathy and age-related macular degeneration. Vitamin C, vitamin E, and

glutathione are important antioxidants that may have potential in the

treatment of retinal diseases caused by oxidative stress. Understanding the

transport characteristics of these antioxidants at the inner blood-retinal barrier

may assist in the design and development of suitable therapy with appropriate

antioxidants for treatment of various retinal diseases. Vitamin C: Vitamin C,

also known as ascorbic acid, exists in the plasma mostly in the oxidized form

dehydroascorbic acid (DHA). The influx permeability rate of DHA is ~40-

fold greater than that of the reduced form ascorbic acid [16]. After entering

the cells, DHA is reduced to generate ascorbic acid. The facilitative glucose

transporter GLUT1 (SLC2A1) is responsible for cellular uptake of DHA, and

this transporter is expressed on both the luminal and abluminal membranes of

the endothelial cells that constitute the inner blood-retinal barrier [16,17].

The primary function of GLUT1 is to transport glucose from blood into

retina. The Michaelis constant for GLUT1 for the transport of glucose is 5-8

mM, which is similar to the physiological plasma concentration of glucose

(~5 mM). Therefore, GLUT1 is not completely saturated with its physiologic

substrate in vivo. Transfer of DHA from blood into retina may occur via

GLUT1 to a great extent even in the presence of normal physiologic levels of

glucose in blood. The fact that GLUT1 is responsible for the delivery of

glucose and DHA to the retina across the inner blood-retinal barrier is very

relevant to diabetic retinopathy. Since plasma levels of glucose rise markedly

in untreated diabetes, the transfer of DHA from blood to retina via GLUT1

may be impaired significantly in diabetes. The resultant deficiency of

antioxidant machinery may contribute to the pathologic sequelae associated

with diabetic retinopathy [18]. Vitamin E: Vitamin E has preventive and

therapeutic effects in human retinopathies. Among the members of the

vitamin E family, -tocopherol has the highest biologic activity, and is


exclusively associated with high-density lipoprotein (HDL) in blood [19].

Uptake of HDL-associated -tocopherol into TR-iBRB cells is most likely

mediated by scavenger receptor class B type I (SR-BI) [20]. Immunostaining

of SR-BI is observed in rat retinal capillaries [20]. SR-BI at the inner blood-

retinal barrier provides an efficient pathway for the supply of -tocopherol to

retina from blood. Cystine: Glutathione is a major antioxidant in the retina. It

is a tripeptide ( -Glu-Cys-Gly). This peptide is synthesized in mammalian

cells using the component amino acids. Intracellular cysteine is low

compared to the other two amino acids, and consequently cysteine represents

the rate-limiting amino acid for glutathione synthesis. Cysteine is present in

plasma predominantly in the oxidized form cystine. Uptake of cystine in

mammalian cells occurs mostly via an amino acid exchanger known as

cystine-glutamate exchanger. It is a Na+-independent process in which influx

of cystine into cells is coupled to efflux of glutamate from the cells. The

transport protein identified as xCT (SLC7A11) is responsible for the process.

xCT is expressed in TR-iBRB cells [21]. The expression and activity of xCT

in these cells is regulated in response to intracellular levels of glutathione.

When the cellular levels of glutathione are depleted by treatment with

diethylmaleate, the expression of xCT is upregulated to facilitate glutathione

synthesis [21,22]. xCT at the inner blood-retinal barrier may thus be an

important determinant of glutathione homeostasis in the retina.

Miscellaneous protective compounds

Compounds such as creatine, carnitine, acetylcarnitine, and taurine are

biologically relevant to retina due to their role in energy homeostasis, fatty acid

oxidation, and calcium signaling. Creatine: Creatine plays a vital role in the

storage and transmission of phosphate-bound energy in retina. The Na+- and

Cl--dependent creatine transporter (CRT, SLC6A8) plays a role in the influx of

creatine into retina at the inner blood-retinal barrier. CRT is localized on both

the luminal and abluminal membranes of rat retinal capillary endothelial cells

[23]. CRT expressed on the luminal membrane would mediate creatine supply

to retina; but the function of CRT on the abluminal membrane is not yet

known. Creatine supplementation into retina is a potentially promising

treatment for gyrate atrophy of the choroid and retina with hyperornithinemia.

However, CRT at the inner blood-retinal barrier is almost saturated by plasma

creatine (140-600 M in mice and rats), since the Michaelis constant for

creatine uptake in TR-iBRB cells (~15 M) is much lower than these plasma

concentrations [23]. The development of drugs which increase the density of

CRT on the luminal membrane and/or CRT transport activity at the inner

blood-retinal barrier is needed for rational creatine therapy of the gyrate


atrophy. Carnitine and acetylcarnitine: Carnitine has multiple roles in

mammalian cells, even though its primary and most recognized function is to

promote fatty acid oxidation for energy production. Acetylcarnitine is effective in

improving visual function in patients with early age-related macular

degeneration. Uptake of carnitine and acetylcarnitine in TR-iBRB cells occurs via

the Na+-dependent organic cation/carnitine transporter 2 (OCTN2, SLC22A5).

OCTN2 is expressed in isolated rat retinal vascular endothelial cells [24]. The

Michaelis constant for the transport of carnitine and acetylcarnitine via OCTN2

in TR-iBRB cells is ~30 M, a value similar to the physiological levels of these

compounds in plasma (carnitine, ~50 M; acetylcarnitine, ~20 M) [24].

Exogenous administration of carnitine and acetylcarnitine would therefore be

able to increase the retinal levels of these protective compounds through OCTN2-

mediated transfer across the inner blood-retinal barrier. Taurine: Taurine, the

most abundant free amino acid in retina, functions as an osmolyte to regulate

cellular volume under altered osmotic conditions. It also has antioxidant

properties and ability to modulate calcium signaling. The Na+- and Cl--dependent

taurine transporter (TAUT, SLC6A6) at the inner blood-retinal barrier mediates

taurine transport from blood to the retina [25]. Since the Michaelis constant for

taurine uptake by TR-iBRB cells (~20 M) is several-fold smaller than the

plasma taurine concentration (100-300 M) in rats, the blood-to-retina taurine

transport appears to be more than 80% saturated by the endogenous taurine under

in vivo conditions [25]. We recently found that TAUT transports -aminobutyric

acid (an inhibitory neurotransmitter) with a lower affinity than taurine; but the

physiologic role of TAUT in the transport of this neurotransmitter in retina is not

readily apparent [26].

Other transport systems

Arginine

Arginine is the precursor for generation of nitric oxide via nitric oxide

synthases. Nitric oxide not only regulates vascular tone and blood flow but

also is a critical component in a variety of cell signaling pathways. It is also

an important determinant in the progression of retinal pathology in diseases

such as diabetic retinopathy and glaucoma. Arginine uptake by TR-iBRB

cells is most likely mediated by the Na+-independent cationic amino acid

transporter 1 CAT1 (SLC7A1) [27]. CAT1 is expressed in retinal capillary

endothelial cells [27]. Since nitric oxide synthesis depends on extracellular

arginine, the function of CAT1 in the delivery of arginine into retina across

the inner blood-retinal barrier may represent a rate-limiting step in nitric

oxide production in retina. Since CAT1 also transports a variety of arginine-


and lysine-based inhibitors of nitric oxide synthases, this transporter at the

inner blood-retinal barrier can be exploited for delivery of such compounds

into retina in the treatment of specific retinal diseases associated with

overproduction of nitric oxide (e.g., inflammation). Lactic acid: Lactic acid

is an important energy source for retinal neurons. Uptake of lactic acid in TR-

iBRB cells occurs via the H+-coupled monocarboxylate transporter 1 MCT1

(SLC16A1) [28]. MCT1 is localized in both the luminal and abluminal

membranes of retinal capillary endothelial cells [29]. A number of

monocarboxylates and monocarboxylic drugs inhibit lactic acid uptake in

TR-iBRB cells. For example, salicylate and valproate competitively inhibit

this process [28]. Therefore, MCT1 has potential for the delivery of

monocarboxylic drugs into retina. Folates: Tetrahydrofolate plays an

essential role 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. Deficiency of folate increases

cellular levels of homocysteine, which has a wide variety of detrimental

effects. Folate deficiency also causes visual dysfunction. Folate in the plasma

of most mammals exists predominantly as the methyl derivative of the

reduced folate, namely N5-methyltetrahydrofolate (MTF). Reduced folate

carrier 1 (RFC1, SLC19A1) mediates MTF uptake by TR-iBRB cells [30].

This process is inhibited by methotrexate and formyltetrahydrofolate. RFC1

mRNA is expressed abundantly in freshly isolated rat retinal endothelial

cells. Methotrexate is widely used as a chemotherapeutic agent in the

treatment of cancer and also as an immunosuppressant in the treatment of

rheumatoid arthritis. When present in circulation, this drug is most likely

transported into retina via RFC1, consequently interfering with the entry of

the physiologic substrate MTF into retina. Choline: Choline is an important

cell membrane constituent in the form of phosphatidylcholine and

sphingomyelin. It is also a precursor of the neurotransmitter acetylcholine.

Choline uptake by TR-iBRB cells is Na+-independent and potential-

dependent, indicating that a specific carrier exists at the inner blood-retinal

barrier for the transfer of this important nutrient into retina [31]. The features

of this uptake process are distinct from those of choline uptake mediated by

other known organic cation transporters. Even though it is clear that a

specific organic cation transporter is responsible for the transfer of choline

into retina across the inner blood-retinal barrier, the molecular identity of the

transporter remains to be established. In general, organic cation transporters

exhibit broad substrate selectivity. Therefore, the transporter responsible for

choline transfer across the inner blood-retinal barrier also has potential for

delivery of specific organic cationic drugs into retina. Glycine: Glycine plays

a pivotal role in neurotransmission and in the biosynthesis of creatine and


glutathione in retina. It is also a co-agonist for the N-methyl-D-aspartate

(NMDA) receptor. In vivo and in vitro studies have demonstrated that the

Na+- and Cl--dependent glycine transporter 1 GlyT1 (SLC6A9) most likely

mediates the blood-to-retina transport of glycine across the inner blood-

retinal barrier [32]. Sarcosine, a methyl-substituted glycine, is also a substrate

for GlyT1, and thus inhibits GlyT1-mediated cellular entry of glycine.

Sarcosine also functions as a co-agonist for the NMDA receptor. Therefore,

sarcosine has potential to modulate the activity of NMDA receptor by

elevating extracellular levels of the co-agonist glycine through inhibition of

glycine uptake via GlyT1 and also by acting directly as a co-agonist. This

glycine analog is currently under investigation for treatment of schizophrenia

as a means to activate the NMDA receptor. Since the NMDA receptor

mediates the signaling of the neurotransmitter glutamate in retina as it does in

central nervous system, the potential of GlyT1 as a delivery system for

glycine and sarcosine into retina may be of clinical significance.

Efflux transporters at the inner blood-retinal barrier

Because the retina produces various metabolites and neurotoxic

compounds, there must be mechanisms at the blood-retinal barrier to eliminate

such compounds as a means to protect the retina from unwanted harmful

effects. Effective efflux transport systems operate in other cell types, which are

located strategically in different tissues to eliminate drugs and other xenobiotics

from the body (e.g., intestine, liver, kidney, blood-brain barrier, placenta).

While such efflux transport systems undoubtedly provide protection against

potentially toxic xenobiotics, the transport systems also pose a significant

problem for effective delivery of therapeutically active drugs if these drugs are

recognized as substrates by the transport systems. Since the efflux transport

systems actively remove their substrates from the cells, the presence of such

transport systems at the blood-retinal barriers would effectively interfere with

the delivery of therapeutic drugs into retina. Therefore, it is important to

identify the efflux transport systems at these barriers and elucidate their

substrate selectivity in terms of various drugs that are of potential use for the

treatment of retinal diseases. Understanding the retina-to-blood efflux transport

of drugs across the inner blood-retinal barrier will provide important

information about the efficacy of drug delivery to the retina.

The retina-to-blood efflux transport of anionic drugs from

vitreous humor/retina across the inner blood-retinal barrier

Many clinically important drugs including antibiotics, anti-tumor drugs,

anti-HIV therapeutics, and anti-inflammatory agents are organic anions. The


limited distribution of -lactam antibiotics in the vitreous humor/retina after

systemic administration is problematic, resulting in reduced efficacy in the

treatment of bacterial endophthalmitis [33]. 6-Mercaptopurine (6-MP) is

frequently used as an anti-cancer drug in patients with childhood acute

lymphoblastic leukemia. Relapse of childhood acute lymphoblastic leukemia

involving eye is a rare but challenging problem. This is probably due to the

restricted distribution of 6-MP in the eye [34]. One possible factor in the

restricted drug distribution in the retina/eye is the retina-to-blood efflux

transport of such anionic drugs across the blood-retinal barriers. In support of

this notion, the transport of fluorescein, an organic anion, in the vitreous-to-

blood direction is more than 100-fold greater than that in the blood-to-

vitreous direction in humans [35]. Betz and Goldstein demonstrated that the

uptake of p-aminohippuric acid (PAH), a well-known organic anion, by

isolated retinal capillaries was greater than that of extracellular marker,

sucrose, and that the uptake was inhibited by fluorescein [36].

We used microdialysis to carry out in vivo evaluation of vitreous/retina-to-

blood efflux transport in rats and to determine the efflux transport of organic

anions across the blood-retinal barriers. 6-MP, PAH, and the -lactam

antibiotic benzylpenicillin (PCG) were injected with D-mannitol, a bulk flow

marker, into the vitreous humor of the rat eye, and a microdialysis probe was

placed in the vitreous humor [37]. PCG, 6-MP, PAH and D-mannitol are

biexponentially eliminated from the vitrous humor after vitreous bolus

injection. The elimination rate constant of PCG, 6-MP, and PAH during the

terminal phase was about 2-fold greater than that of D-mannitol. This efflux

transport was reduced in the retina in the presence of probenecid, PAH, and

PCG, relatively specific substrates of organic anion transporter (Oat) 3

(SLC22A8) [38]. Oat3 is localized on the abluminal membrane of retinal

capillary endothelial cells [37]. Thus, Oat3 is involved in the uptake of PCG

and 6-MP across the abluminal membrane of retinal capillary endothelial cells

and contributes to the efflux transport of PCG and 6-MP from vitreous

humor/retina into blood across the inner blood-retinal barrier. This process

would be a critical factor in the restricted distribution of anionic drugs in retina.

Some -lactam antibiotics are substrates for organic anion transporter

polypeptide (oatp) 1a4 (Slco1a4; oatp2), which is present at the inner blood-

retinal barrier in the rat [39,40]. We have recently reported that estradiol 17 -

glucuronide undergoes efflux transport from retina via oatp1a4 at the blood-

retinal barriers. Taken together, oatp1a4 could also be involved in the clearance

of anionic -lactam antibiotics at the inner blood-retinal barrier [41].

The retina-to-blood efflux transport of drugs consists of two steps, i.e.,

influx across the abluminal membrane from retina into retinal capillary


endothelial cells and subsequent efflux across the luminal membrane from

endothelial cells into blood. An ATP-dependent efflux pump for such anionic

drugs is likely to exist on the luminal membrane to carry out the efflux process.

ATP-binding cassette (ABC) transporter C 4 (multidrug resistance-associated

protein, MRP4) is a promising candidate for the transport of these anionic

drugs. Indeed, MRP4 transports several anionic drugs such as PAH, and also 6-

MP and -lactam antibiotics such as PCG [42]. We have demonstrated that

MRP4 (ABCC4) mRNA is expressed abundantly in isolated mouse and rat

retinal vascular endothelial cells [3,43]. It has been demonstrated that MRP4 is

localized on the luminal membrane of human brain capillary endothelial cells

[44], although the protein localization of MRP4 at inner blood-retinal barrier is

yet to be demonstrated. These findings imply that MRP4 acts as an active

efflux transporter for anionic drugs at the inner blood-retinal barrier and

decreases the blood-to-retina transfer of -lactam antibiotics and 6-MP. In

addition to MRP4, ABCC3 (MRP3) and ABCC6 (MRP6) mRNAs are also

expressed abundantly in isolated mouse retinal vascular endothelial cells [43].

Other transporters involved in the retina-to-blood efflux transport

at the inner blood-retinal barrier

We have found that Oatp1c1 (Slco1c1/Oatp14) mRNA is highly

expressed in isolated rat retinal endothelial cells [45]. Oatp1c1 transports

estradiol 17 -glucuronide as is the case with Oatp1a4. On the other hand,

Oatp1c1 does not have high affinity for digoxin [46], a specific substrate of

Oatp1a4. This suggests that Oatp1c1 and Oatp1a4 play distinct roles in the

retina-to-blood efflux transport in terms of the specificity of the drugs and

xenobiotics that the two transporters handle. Further studies are needed to

clarify the individual contribution of Oatp1c1 and Oatp1a4 to the efflux of

specific anionic drugs across the inner blood-retinal barrier. Other members

belonging to the ATP-dependent family of efflux transporters such as ABCA,

ABCB, and ABCG could play a role in restricting the distribution of

endobiotics and xenobiotics, including therapeutic agents, in retina. ABCB1

(P-glycoprotein, P-gp) is localized on the luminal membrane of retinal

capillary endothelial cells [4]. The active efflux transport function of P-gp at

the inner blood-retinal barrier could lower the blood-to-retina permeability of

its substrates. For example, cyclosporin A, a substrate of P-gp, was not

detected in the intraocular tissues of cyclosporine A-treated rabbits, although

the blood level of cyclosporine A was within the therapeutic window [47].

TR-iBRB cells express P-gp, and rhodamine 123 accumulation in TR-iBRB

cells is enhanced in the presence of inhibitors of P-gp [5]. ABCG2 (breast

cancer resistance protein BCRP/MXR/ABCP) is also expressed on the


luminal membrane of the inner blood-retinal barrier [48]. ABCG2 recognizes

as its substrates not only drugs such as mitoxantrone and doxorubicin, but

also photosensitive toxins such as pheophorbide a, a chlorophyll-derived

dietary phototoxin related to porphyrin. In vitro studies have demonstrated

that ABCG2 is involved in the excretion of pheophorbide a from TR-iBRB

cells [48]. Because retina is subject to high levels of cumulative irradiation,

ABCG2 may protect this tissue from the light-induced damage caused by a

variety of phototoxic compounds including porphyrins. ABCA transporters

play an essential role in the efflux of endogenous lipids such as sterols,

phospholipids and retinoids from cells. We recently demonstrated that

ABCA3 and ABCA9 mRNAs are highly expressed in isolated mouse retinal

vascular endothelial cells [43]. Thus, the ABC transporters collectively

exhibit a very broad range of substrate selectivity, and work cooperatively at

the inner blood-retinal barrier to provide an effective protective mechanism

for the retina by restricting the entry of potentially harmful chemicals into

retina. But at the same time, this mechanism also poses a significant problem

in the delivery of specific drugs into retina for therapeutic purposes. A clear

understanding of the various influx and efflux transporters that are expressed

at the inner blood-retinal barrier and their detailed substrate selectivity is

necessary to determine whether or not a given drug would enter retina from

blood in therapeutically relevant concentrations.

Influx transporters at the outer blood-retinal barrier

RPE cells constitute the outer blood-retinal barrier. These cells form a

tight monolayer, separating the neural retina from the choroidal blood

circulation. The endothelial cells of the choroidal capillaries are highly

fenestrated and thus do not function as a barrier for the transfer of nutrients or

xenobiotics. Therefore, it is the monolayer of the RPE cells that provides the

barrier function. Since one of the important biologic roles of RPE is to

provide essential nutrients to photoreceptors, this cell expresses a multitude

of transporters, both on the choroid-facing basolateral membrane and the

photoreceptor-facing apical membrane. Even though RPE is a polarized cell

similar to the epithelial cells of the intestine and kidney, the distribution of

various transporters in the apical versus the basolateral membrane in RPE is

strikingly different from that in the other two cell types. For example, the

Na+/K+ pump is localized in the blood-facing basolateral membrane in

intestinal and renal epithelial cells to mediate the absorption of Na+ from the

intestinal or kidney tubular lumen into blood. In contrast, the pump is located

not in the blood-facing basolateral membrane but in the apical membrane on

the opposite side in RPE. This differential localization is necessary to


mediate the transfer of Na+ from blood into neural retina. This is true for

several other transporters as well. The “reverse polarity” in RPE is not all that

surprising, considering the fact that the RPE cells transport nutrients from

blood into neural retina whereas the intestinal and renal epithelial cells

transport nutrients into blood. Several different approaches have been used to

investigate transport processes in RPE and to identify the transporters that are

responsible for these processes. Apical membrane vesicles from RPE can be

isolated easily, provided the amount of available tissue is large enough (e.g.,

bovine eyes). These membrane vesicles are ideal to study the function of

apical membrane transporters in a cell-free system. Isolated RPE /choroid

preparations have also been used with Ussing chambers to investigate the

directional movement of nutrients. Alternatively, RPE cells can be isolated

and used directly for transport studies. However, the cells may not polarize

well if cultured on impermeable plastic supports. But, when cultured on

permeable membrane supports, the cells do polarize with the basolateral

membrane in contact with the membrane support and apical membrane away

from the membrane support. This culture system is suitable not only for

immunocytochemical localization of the transporters present in the apical

versus basolateral membranes but also for studies of vectorial transfer of

nutrients or xenobiotics in the apical-to-basolateral or basolateral-to-apical

direction. A considerable amount of work on the transport characteristics of

RPE has also been carried out using the ARPE-19 cell line. This cell line also

polarizes when cultured on permeable membrane supports [50]. Recently,

primary cultures of RPE cells from human fetal eyes have also been shown to

form polarized monolayers on permeable membrane supports under specific

culture conditions [51]. With the use of these various approaches, a great deal

of information is now available on the identity and characteristics of

transporters in the apical and basolateral membranes of the RPE cells to

understand the role of various transporters in the handling of xenobiotics and

therapeutic drugs at the outer blood-retinal barrier.

Transporter-mediated drug delivery across the outer blood-

retinal barrier

Amino acid mimetic drugs

L-DOPA and amino acid mustards represent examples of amino acid

mimetic drugs. These compounds are recognized as substrates by the Na+-

independent amino acid transport system known as System L. At the

molecular level, System L is comprised of several isoforms. RPE cells

express mRNAs for LAT1 (SLC7A5) and LAT2 (SLC7A8) [52]. Both LAT1


and LAT2 are obligatory amino acid exchangers, meaning that influx of one

amino acid substrate into cells is coupled obligatorily to efflux of some other

amino acid substrate. LAT1 exhibits high affinity towards its substrates

whereas LAT2 shows relatively much lower affinity. In addition, LAT1

prefers bulky neutral amino acids and also recognizes certain D-amino acids;

in contrast, LAT2 has much broader substrate selectivity, though its

substrates also must be neutral amino acids. Furthermore, LAT2 does not

transport D-amino acids. The high affinity transport of D-serine by LAT1

may be of clinical signficance because this D-amino acid functions as a

potent co-agonist of the NMDA receptor. Functional studies have confirmed

the expression of LAT2 in RPE [53-55]. Interestingly, the transport of

leucine, a bulky neutral amino acid, occurs in the apical-to-basolateral

direction in RPE [54,55]. This does not mean however that LAT2 functions

exclusively in efflux of amino acids from retina into blood across the outer

blood-retinal barrier. Since LAT2 is an amino acid exchanger, the efflux of

leucine across the cell has to be coupled to influx of certain other amino acids

in the blood-to-retina direction. LAT2 is expressed exclusively in the

basolateral membrane of intestinal and kidney epithelial cells [56]. Recently,

Yamamoto et al [57] found that LAT1 and LAT2 are expressed at the

functional level in ARPE-19 cells. At the outer blood-retinal barrier, the

amino acid flux is primarily from the circulating blood into retina. The

location of LAT2 in RPE seems to be analogous to what is found in the

intestinal and renal epithelial cells mediating the efflux of amino acids from

the cells (i.e., on the apical membrane of RPE facing the neural retina, but on

the basolateral membrane of intestinal and renal epithelial cells facing the

blood) [56]. LAT1 may be expressed in RPE on the basolateral membrane,

although this remains yet to be established. RPE also expresses mRNA for

y+LAT1 (SLC7A7) [52], which transports cationic amino acids such as

arginine and lysine in a Na+-independent manner and neutral amino acids in a

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 a functional coupling between LAT1/LAT2 and y+LAT1 in the

vectorial transfer of amino acid mimetic drugs across RPE, but the polarity of

the distribution of these transporters needs to be established to make a

rational prediction of their potential in the delivery of such drugs into retina.

Antioxidants and other protective compounds

We have recently reviewed the transport of vitamin C in the retina and

the transporters involved in the process [58]. Since the reduced form of the


vitamin (known as ascorbic acid) is transported via the Na+-coupled

transporters SVCT1 (SLC23A1) and SVCT2 (SLC23A2) whereas the

oxidized form of the vitamin (known as dehydroascorbic acid, DHA) is

transported via the facilitative glucose transporter GLUT1 (SLC2A1), the

expression and localization of these transporters in RPE would determine the

molecular species of the vitamin that is transported, the direction in which the

transfer of the vitamin occurs, and the identity of the transporters that are

responsible for the process, at the outer blood-retinal barrier. GLUT1 is

expressed abundantly in RPE, and is present both at the apical membrane and

basolateral membrane [17]. Of the two Na+-coupled vitamin C transporters,

RPE expresses predominantly SVCT2 [58]. Cultured RPE cells as well as

RPE cell lines take up ascorbic acid in a Na+-dependent manner with high

affinity [59-61]. There is no information available on the polarized

expression of SVCT2 in RPE, but the functional studies have shown that the

Na+-dependent transport of ascorbic acid occurs predominantly at the apical

membrane [61]. Based on the expression pattern of GLUT1 and the Na+-

coupled ascorbic acid transport in RPE, it seems that vitamin C enters RPE

cells from choroidal blood primarily in the form of DHA via GLUT1 (Fig.

3A). Once inside the cell, DHA is reduced into ascorbic acid for subsequent

use in the cell as an antioxidant. Vitamin C also enters RPE cells from

subretinal space via SVCT2 at the apical membrane. The expression of

GLUT1 also at the apical membrane suggests that DHA that comes into RPE

from choroidal blood via GLUT1 at the basolateral membrane, and which is

generated inside the cells from ascorbic acid during antioxidant reactions

may be delivered into subretinal space via the apical membrane GLUT1 for

use by photoreceptors and other retinal cells. Cystine: Cystine, which plays a

critical role in the maintenance of cellular levels of glutathione, is transported

into mammalian cells by the amino acid transporter xc-. This transporter

consists of the “transporter proper” xCT and the chaperone 4F2hc. Functional

and immunocytochemical studies have shown that RPE expresses this

transporter [62,63], and that the transporter may play a role in RPE cell

proliferation [63]. The expression of the transporter is upregulated in RPE

under conditions of increased oxidative stress, indicating a protective role of

this transporter as an antioxidant mechanism through glutathione [63,64].

ARPE-19 cells also express xc- robustly; the Michaelis constant for cystine

for transport via xc- is in the range of 80-100 M [65]. Expression of the

transporter in these cells is markedly upregulated by the transactivator protein

Tat encoded by HIV-1 genome [65]. Even though these studies have

demonstrated unequivocally the expression of the transporter in RPE, there is

no information available on the location of the transporter in the apical versus

the basolateral membrane. Creatine: Recently we were comparing the gene


Figure 3. Models for the transport of vitamin C (A), lactate (B), and folate (C) in RPE

that constitutes the outer blood-retinal barrier. GLUT1, facilitative glucose transporter

1; SVCT2, sodium-coupled vitamin C transporter 2; DHA, dehydroascorbic acid; AA,

ascorbic acid; MCT1, monocarboxylate transporter 1; MCT3, monocarboxylate

transporter 3; SMCT1, sodium-coupled monocarboxylate transporter 1; FR , folate

receptor ; PCFT, proton-coupled folate transporter; RFC, reduced folate carrier; F,

folate and its analogs such as N5-methyltetrahydrofolate and methotrexate.

expression pattern between control ARPE-19 cells and ARPE-19 cells transfected

with HIV-1 Tat cDNA (unpublished results) using microarray. These studies

revealed that ARPE-19 cells express the creatine transporter (SLC6A8) and that

the expression is upregulated by HIV-1 Tat. The uptake of creatine in control

ARPE-19 cells is Na+- and Cl- -dependent, and saturable. The Michaelis constant

for the transport process is 36 6 M. The Na+- and Cl- -activation kinetics of

creatine uptake indicated that the Na+:Cl-:creatine stoichiometry is 2:1:1. The

expression of HIV-1 Tat in these cells enhanced creatine uptake, and the process

was associated with an increase in the steady-state levels of the transporter

mRNA as evident from RT-PCR and Northern blot, transporter protein as evident

from western blot, and transport activity as evident from the increase in maximal

velocity. There was no change in substrate affinity nor in Na+:Cl-:creatine


stoichiometry. We have not yet examined the expression of the transporter in

primary RPE cells and we also do not know whether or not the transporter is

differentially expressed in the apical versus the basolateral membrane. Carnitine

and acetylcarnitine: Carnitine is transported at least by two different transport

systems, namely the Na+-dependent carnitine transporter OCTN2 (SLC22A5)

[66,67] and the Na+- and Cl- -dependent amino acid transporter ATB0,+

(SLC6A14) [68]. Uptake studies with ARPE-19 cells have provided evidence for

transport of carnitine via both transporters (unpublished results). Saturation

kinetics of Na+-dependent carnitine uptake showed the presence of two distinct

transport systems responsible for the observed uptake, one with high affinity

(Michaelis constant, 3.2 0.4 M) and the other with low affinity (Michaelis

constant, 655 87 M). Based on the known affinities of OCTN2 and ATB0,+,

the high affinity uptake is likely to be mediated by OCTN2 and the low affinity

uptake by ATB0,+. At micromolar concentrations, carnitine uptake occurs

predominantly via OCTN2. This high affinity uptake is inhibited by

acetylcarnitine, suggesting that OCTN2 accepts this carnitine derivative as a

substrate. In addition to carnitine and its acylderivatives, several other cationic

and zwitterionic drugs are transportable substrates for OCTN2. This includes -

lactam antibiotics [69], tetraethylammonium, pyrilamine, quinidine, verapamil,

and valproate [70]. Similarly, SLC6A14 is capable of transporting a wide variety

of drugs and prodrugs [71], including nitric oxide synthase inhibitors [72] and

amino acid derivatives of antiviral agents such as valacyclovir [73] and

valgancicolovir [74]. These data show that RPE expresses OCTN2 and ATB0,+

with the ability to transport carnitine, acylcarnitines, and a multitude of drugs, but

the polarity of their expression in the apical versus basolateral membrane remains

to be determined. Taurine: Several studies have documented the expression of

the Na+/Cl- -coupled taurine transporter (SLC6A6) in RPE [75-79]. There is

unequivocal evidence for the presence of the transporter in the apical membrane

of this cell. Isolated apical membrane vesicles from bovine RPE demonstrate

robust Na+/Cl- -coupled taurine uptake [75,76]. There is also evidence for

potential contribution of a -aminobutyrate transporter to the transport of taurine

at this membrane [76]. RPE cell lines are highly active in taurine uptake [77-79],

and the expression and activity of the transporter are subject to regulation by

nitric oxide [78] and by changes in extracellular osmolality [79].

Monocarboxylic drugs

Endogenous monocarboxylates such as lactate, pyruvate, and ketone

bodies ( -hydroxybutyrate and acetoacetate) are transported in mammalian


cells via two distinct classes of transporters, namely the H+-coupled

electroneutral monocarboxylate transporters belonging to the SLC16 gene

family (MCTs) and the Na+-coupled electrogenic monocarboxylate transporters

belonging to the SLC5 gene family (SMCTs). RPE expresses MCT1

(SLC16A1), MCT3 (SLC16A8), and SMCT1 (SLC5A8). MCT1 is expressed

exclusively in the apical membrane of RPE whereas MCT3 is expressed

exclusively in the basolateral membrane (Fig. 3B), both in the form of their

heterodimeric complexes with the cell surface glycoprotein CD147 [80-82].

Since there is no significant H+ gradient across the apical membrane or the

basolateral membrane in RPE in vivo, MCT1 and MCT3 are able to transport

their endogenous substrates either into the cell or out of the cell, depending on

the concentration gradients for the substrates. Several monocarboxylic drugs

have been shown to be substrates for MCT1; this includes foscarnet, salicylate,

benzoate, and a prodrug of gabapentin [83,84]. -Hydroxybutyrate, an

endogenous monocarboxylate as well as a therapeutic drug, is also a substrate

for MCT1 [84]. SMCT1 is expressed only in the basolateral membrane of RPE

[85]. Since the transport process mediated by SMCT1 is Na+-coupled and

electrogenic [86], the substrates of SMCT1 are actively transported into RPE

from choroidal blood (Fig. 3B). The endogenous monocarboxylates and the

monocarboxylic drugs that are recognized by SMCT1 as substrates include

lactate, pyruvate, short-chain fatty acids such as propionate and butyrate [86],

the B-complex vitamin nicotinate [87], ketone bodies [88], -hydroxybutyrate

[89], benzoate, salicylate, 5-aminosalicylate [90], and 3-bromopyruvate [91].

Recent studies have shown that the cysteine prodrug L-2-oxothiazolidine

carboxylate is a high affinity substrate for SMCT1 [91a], indicating that

SMCT1 in the RPE basolateral membrane can be exploited to deliver this drug

into cells to increase intracellular glutathione levels as a means to reduce

oxidative stress. Interestingly, nonsteroidal anti-inflammatory 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 [92].

Folate and folate analogs

There are at least three transport proteins that play a role in the uptake of

folate and its analogs in mammalian cells. These are the reduced folate carrier

(RFC, SLC19A1), folate receptor , and the proton-coupled folate transporter

(PCFT, SLC49A1) [93]. All three proteins are expressed in RPE [94-97]. RPE

is capable of transcellular transfer of folate, indicating that the folate transport


proteins must be expressed in this cell in a polarized manner [98]. Folate

receptor is expressed exclusively in the choroid-facing basolateral membrane

whereas the reduced folate carrier is expressed exclusively in the apical

membrane (Fig. 3C) [96]. Since RPE constitutes the outer blood-retinal barrier,

the essential vitamin folate must pass through the cell in the blood-to-retina

direction. Folate exists predominantly in the form of N5-methyltetrahydrofolate

in plasma, and therefore this form of folate is the principal substrate for the

folate transport proteins in RPE. Since the transfer across the basolateral

membrane is the first step in the movement of folates across RPE from blood

into retina, folate receptor must participate in this first step. However, the

folate receptor -mediated entry into cells results in the delivery of folates into

endosomes. Therefore, an additional step is needed to deliver folates from the

endosomes into cytoplasm. We postulated several years ago that a hitherto

unidentified transport system is necessary to mediate this process [99]. The

recently cloned proton-coupled folate transporter PCFT has all the features

necessary for the delivery of folates from the endosomes into cytoplasm [100].

Since the endosomal compartment is acidic, there exists a H+ gradient across

the endosomal membrane in the endosome-to-cytoplasm direction. Such a

gradient would be ideal to drive PCFT. PCFT is indeed expressed in RPE [97],

but its localization in the basolateral membrane has not been demonstrated. We

strongly believe that PCFT is expressed in RPE in a polarized manner with

exclusive localization in the basolateral membrane, colocalizing with folate

receptor . The transfer of folates across this membrane would involve a

functional coupling between the folate receptor and PCFT (Fig. 3C). Folate will

bind to the receptor first to initiate endocytosis. Because of the colocalization,

endocytosis will result in the transfer of the receptor-folate complex as well as

PCFT into endosomes. When the endosomes are acidified subsequently, folate

will dissociate from the receptor and then get transferred into the cytoplasm via

PCFT. This process will be energized by the transmembrane H+ gradient that

exists across the endosomal membrane. The reduced folate carrier in the apical

membrane will then facilitate the delivery of folate from the cytoplasm into the

subretinal space, thus completing the transcellular transfer. Folate analogs that

serve as antifolates (e.g., methotrexate, pemetrexed) can be delivered into

neural retina across the outer blood-retinal barrier via the concerted actions of

the three folate transport proteins. Recently, Zhao et al have provided evidence

for a role for PCFT in folate receptor-mediated endocytosis [101]. With

electron microscopic analysis, we have shown that folate receptor and PCFT

colocalize in Muller cells on the plasma membrane as well as on the endosomal

membrane [102]. Muller cells take up folates using these two transport


proteins. Such studies have not yet been done with RPE, but we hypothesize

that PCFT colocalizes with folate receptor in the basolateral membrane and

that the two proteins work together in the delivery of folates from choroidal

circulation into cells with the participation of endosomes as an intermediate. Biotin, pantothenate, and lipoic acid

Biotin and pantothenate are also water-soluble vitamins that exist predominantly in anionic form. As any other mammalian cell, RPE and other retinal cells require these vitamins for their biological function. These vitamins are transported via SMVT, a Na+-coupled mutltivitamin transporter. SMVT also accepts lipoic acid as a substrate. Lipoic acid is a potent antioxidant, and it protects RPE cells from oxidative stress [103,104]. The expression of SMVT in RPE in intact retina has not been studied, but the transporter is expressed in the human RPE cell line ARPE-19 [105]. In addition to the normal physiologic role in the cellular uptake of biotin, pantothenate, and lipoic acid, SMVT also has potential as a delivery system for a variety of prodrugs [105,106].

Organic cationic drugs

Endogenous and exogenous organic cations are transported into

mammalian cells via a variety of organic cation transporters [107]. One of

these transporters is OCT3 (SLC22A3) that transports its substrates in an

electrogenic manner. This transporter is expressed in RPE cells [108]. The

exact location of the transporter in RPE (apical versus basolateral) is not

known, but its substrates of pharmacological and therapeutic significance

include prazocin ( -adrenoceptor antagonist), clonidine ( -adrenoceptor

agonist), cimetidine (histamine H1 receptor antagonist), verapamil (calcium

channel blocker), imipramine and desipramine (antidepressants), quinine

(antimalarial drug), and nicotine and methylenedioxymethamphetamine

(addictive drugs) [107]. There is also evidence of a novel organic cation

transporter in RPE cells that has not been characterized at the molecular

level. This transporter transports verapamil, diphenhydramine, pyrilamine,

quinidine, and quinacrine [109]. The same transporter may also transport the

2-adrenergic agonist brimonidine [110].

Vitamin E and carotinoids

The scavenger receptor class B type 1 (SR-BI) and its splice variant SR-

BII are expressed in RPE [111,112], and the expression is found both in the

apical and basolateral membranes [112]. In various cell types including the


retinal blood vessel endothelial cell line TR-iBRB, SR-BI is responsible for the

uptake of vitamin E, an antioxidant, either in the free form or when present as a

component of HDL [20,113]. Therefore, it is likely that SR-BI mediates the

uptake of this vitamin from choroidal blood into RPE. Since most of vitamin E

in blood is associated with HDL, SR-BI in RPE does not transport free vitamin

E in vivo but rather the HDL-associated vitamin E. The xanthophylls

carotenoids such as lutein, zeaxanthin, and lycopene play a significant role in

the maintenance of normal vision. These carotenoids are taken up into

differentiated ARPE-19 cells via SR-BI [114], suggesting that a similar

mechanism might operate in vivo in the uptake of these pigments from blood.

Opioid peptides and peptidomimetic drugs

RPE cells express two novel oligopeptide transport systems that handle a

wide variety of endogenous and synthetic opioid peptides [115,116]. Non-

peptide opioids or non-peptide opioid antagonists do not interact with these

transport systems. The two transport systems are called sodium-coupled

oligopeptide transporters SOPT1 and SOPT2. These transport systems are

distinct from the H+-coupled peptide transporters PEPT1 and PEPT2. In

addition to opioid peptides, SOPT1 and SOPT2 also transport other

oligopeptides such as the peptide fragments of HIV-1 Tat (e.g., Tat47-57) and

HIV-1 Rev (e. g., Rev34-50). These transport systems are upregulated in

ARPE-19 cells several-fold by HIV-1 Tat. There is a marked overlap

between SOPT1 and SOPT2 in substrate specificity, but the two transport

systems are distinguishable based on the opposing effects of small peptides

on transport activity. Dipeptides and tripeptides stimulate the activity of

SOPT1 but inhibit the activity of SOPT2. The dipeptide opioid kyotorphin is

not a substrate for these oligopeptide transport systems, but it stimulates the

activity of SOPT1 and inhibits the activity of SOPT2 [116,117]. Peptides

consisting of up to 25 amino acids have been shown to interact with these

transport systems. SOPT1 and SOPT2 hold great potential for the transport of

peptide and peptidomimetic drugs into RPE. The exact location of these

transporters in RPE has not yet been established.

Organic anion transporting polypeptides

This group of transporters mediates Na+-independent transport of a wide

variety of organic compounds such as bile salts, dyes, steroid sulfates and

glucuronides, anionic oligopeptides, digoxin, thyroid hormones, and

prostaglandins. The expression of two of these transporters has been studied

in rat retina [118, 119]. Oatp2, encoded by Slco1a4, is expressed prominently


in the apical membrane of RPE [118]. Oatp-E, encoded by Slco4a1, is

expressed in RPE, but its exact location is not known [119].

Efflux transporters at the outer blood-retinal barrier

RPE cells that constitute the outer blood-retinal barrier play an essential

role not only in the delivery of nutrients to retina but also in the protection of

this tissue from potential toxic effects of endobiotics and xenobiotics when

present in systemic circulation. This protective role of RPE involves

prevention of entry of such toxic molecules from choroidal blood into retina.

Two different mechanisms participate in this process. The toxic endobiotics

and xenobiotics in the systemic circulation might gain entry into RPE either

by diffusion or by specific influx transporters in the basolateral membrane.

These molecules can be effluxed out of RPE back into systemic blood via

specific efflux transporters that are expressed in the RPE basolateral

membrane. The second mechanism involves transcellular transfer of

endobiotics and xenobiotics from subretinal space into systemic blood via

concerted actions of influx transporters in the apical membrane and efflux

transporters in the basolateral membrane. While it is certainly true that these

efflux processes play a beneficial role in the protection of retina from

potentially toxic xenobiotics, these processes also pose a major problem in

the delivery of therapeutic drugs to retina. Many of the widely used and

clinically relevant drugs are substrates for the efflux transporters and

therefore such drugs are actively removed from retina through RPE, thus

preventing accumulation of these drugs in retina to therapeutically effective

concentrations. This hurdle can be overcome however if specific inhibitors of

the efflux transporters are co-administered along with the drugs. This

provides a means to prevent the removal of the drugs from retina via the

efflux transporters in RPE.

P-glycoprotein, BCRP, and MRPs

P-glycoprotein, encoded by the MultiDrug Resistance gene MDR1, is

expressed in RPE. It is an ATP-dependent efflux pump; its expression has

been demonstrated in a number of human RPE cell lines (e.g., D407, h1RPE),

but interestingly not in ARPE-19 [120-122]. Several classes of drugs,

including anticancer agents, antibiotics, steroids, and immunosuppressants

are recognized as substrates for P-glycoprotein. Daunomycin, which is used

for the management of proliferative vitreoretinopathy, is a substrate for this

efflux pump. Treatment of patients with proliferative vitreoretinopathy using

daunomycin causes overexpression of P-glycoprotein, thus resulting in


multidrug resistance [123]. This is similar to the multidrug resistance

observed in certain cancers due to overexpression of this efflux pump. The

transporter is expressed more predominantly in the basolateral membrane

facing the choroidal circulation where it can mediate active transfer of its

substrates from RPE into blood [120]. Interestingly, it is also found in the

apical membrane of RPE where its physiologic function remains unknown.

The breast cancer resistance protein BCRP, also known as ABCG2, is

expressed in D407 cells but not in any other RPE cell line [122]. RPE in

intact retina as well as primary RPE cells express BCRP; the transporter

localizes to basolateral membrane of this cell [124]. BCRP is also an efflux

pump energized by ATP hydrolysis. There is clear evidence of expression of

Multidrug Resistance-associated Protein (MRP) functional activity in ARPE-

19 cells and in primary cultures of human RPE [125]. The aldose reductase

inhibitor BAPSG (N[4-(benzoylamino)phenyl sulfonyl]glycine), which has

potential for treatment of diabetic retinopathy, is a substrate for this efflux

process [125]. Among the six different genes coding for MRPs, three (MRP1,

MRP4, and MRP5) are expressed in all human RPE cell lines [122]. MRPs

are also dependent on ATP hydrolysis for active efflux of their substrates.

The expression profile of MRPs in intact retina has not been studied.

Conclusions and perspectives

Even though the neural retina is isolated from systemic circulation via

the inner and outer blood-retinal barriers, the plasma membrane transporters

expressed at these barrier sites can be exploited for delivery of a wide variety

of drugs into retina. An in-depth knowledge of the transporters in the retinal

blood vessel endothelial cells and retinal pigment epithelial cells is an

absolute requirement to appreciate and exploit the full potential of this

approach to deliver therapeutic drugs into the posterior part of the eye for

treatment of retinal diseases. Several factors related to specific characteristics

of these transporters need to be taken into consideration to maximize their

potential use as drug delivery systems. This includes knowledge on relative

affinities of these transporters for endogenous substrates versus therapeutic

agents to minimize competition, transport capacities to maximize drug delivery

for achievement of therapeutically relevant concentrations, and tolerance for

structural alterations in substrates to broaden the scope in the design of drugs

that can be delivered. We have witnessed a remarkable progress in recent years

in the identification and characterization of the transporters in these two cell

types that constitute the blood-retinal barriers. This information will be very

useful for the future design and development of specific drugs that can be

delivered efficiently into retina for therapeutic purposes.


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