Download 1. Transport of drugs across the inner and outer blood

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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
1
Department 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
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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-toretinal 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 bloodretinal 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
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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.
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Influx transporters at the inner blood-retinal barrier
The retinal capillary endothelial cells that constitute the inner bloodretinal 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 Dglucose (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.
Drug delivery to retina
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Figure 2. Efflux and influx transporters expressed in retinal capillary endothelial cells
that constitute the inner blood-retinal barrier. DHA, dihydroascorbic acid; MTF, N5methyltetrahydrofolate.
Transporter-mediated drug delivery across the inner bloodretinal 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
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Masanori Tachikawa et al.
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, phenylglycinemustard 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-
Drug delivery to retina
7
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 lightinduced 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 ~40fold 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
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Masanori Tachikawa et al.
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 bloodretinal 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
Drug delivery to retina
9
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 OCTN2mediated 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-
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Masanori Tachikawa et al.
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 TRiBRB 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 potentialdependent, 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
Drug delivery to retina
11
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 bloodretinal 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
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Masanori Tachikawa et al.
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-toblood direction is more than 100-fold greater than that in the blood-tovitreous 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-toblood 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 bloodretinal barrier in the rat [39,40]. We have recently reported that estradiol 17 glucuronide undergoes efflux transport from retina via oatp1a4 at the bloodretinal 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
Drug delivery to retina
13
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 6MP 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
14
Masanori Tachikawa et al.
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
Drug delivery to retina
15
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 bloodretinal 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
16
Masanori Tachikawa et al.
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
Drug delivery to retina
17
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
18
Masanori Tachikawa et al.
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
Drug delivery to retina
19
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
20
Masanori Tachikawa et al.
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
Drug delivery to retina
21
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
22
Masanori Tachikawa et al.
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 SRBII 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
Drug delivery to retina
23
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]. Nonpeptide 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
24
Masanori Tachikawa et al.
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
Drug delivery to retina
25
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 ARPE19 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.
26
Masanori Tachikawa et al.
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