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Title Page
Drug transport by the blood-aqueous humor barrier of the eye
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Jonghwa Lee and Ryan M. Pelis
Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada
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Running Title Page
Running Title: Drug transport by the blood-aqueous humor barrier
Corresponding Author:
Ryan M. Pelis, Ph.D.
Department of Pharmacology
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Dalhousie University
Sir Charles Tupper Medical Building
5850 College Street, PO Box 15000
Halifax, Nova Scotia, Canada B3H 4R2
#Text pages: 16
#Tables: 2
#Figures: 2
#References: 65
#Words in Abstract: 240
Abbreviations: Apical sodium dependent bile salt transporter, ASBT; Breast cancer resistant
protein, BCRP; Multidrug and toxin extrusion transporter, MATE; Multidrug-resistance
associated protein, MRP; Organic anion transporter (OAT); Organic anion transporting
polypeptide, OATP; Organic cation transporter, OCT; Organic cation transporter novel, OCTN;
Peptide transporter, PEPT; P-glycoprotein, Pgp; Sodium dicarboxylate cotransporter, NaDC;
Sodium taurocholate cotransporter, NTCP
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Abstract
The ocular barriers (cornea, blood-retinal barrier and blood-aqueous humor barrier) make
treating eye diseases with therapeutic drugs challenging. The tight capillary endothelium of the
iris and the ciliary body epithelium form the blood-aqueous humor barrier. The iris and ciliary
body (iris-ciliary body) express a variety of drug transporters in the ATP-binding cassette (ABC)
and solute carrier (SLC) families. Drug transporters in the ABC family that are present in the
iris-ciliary body include P-glycoprotein (Pgp), breast cancer resistant protein (BCRP) and several
present include organic anion transporters (OATs), organic anion transporting polypeptides
(OATPs), bile acid transporters (ASBT and NTCP), organic cation transporters (OCTs, OCTNs
and MATEs) and peptide transporters (PEPTs). Freshly dissected iris-ciliary body preparations
actively accumulate a variety of substrates of SLC drug transporters that are expressed in the
tissue. The ciliary body in vitro supports active transport in the aqueous humor-to-blood
direction of several substrates of OATs and MRPs, consistent with the subcellular localization of
these transporters in the ciliary body epithelium. In vivo data suggest that drug transporters in
the iris-ciliary body reduce the permeation of drugs in the direction of blood-to-aqueous humor,
thereby reducing ocular drug bioavailability, but also, are involved in active drug elimination
from the aqueous humor. A better understanding of the influence on pharmacokinetics of drug
transporters in the blood-aqueous humor barrier should help improve drug delivery and efficacy
in the eye.
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multidrug resistance-associated proteins (MRPs). Drug transporters in the SLC family that are
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Introduction
The eye is a pharmacological sanctuary, making it challenging to treat many ocular
diseases with therapeutic drugs. This is in part due to the presence of the ocular barriers –
cornea, blood-retinal barrier (inner and outer) and blood-aqueous humor barrier (iris-ciliary
body). The resilience of the eye to drug exposure is evident from the number of ocular drug
delivery methods in development and use. Topical, systemic, periocular and intraocular are the
main routes for drug delivery to the eye (Geroski and Edelhauser, 2000). Therapeutic
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concentrations can be achieved with both topical and systemic administration, but often requires
the use of relatively high doses and/or drugs with sufficient hydrophobicity to efficiently cross
the ocular barriers and cornea (Barar, et al., 2008;Gaudana, et al., 2010). Although topical eye
drop administration is the least invasive, only a small fraction of the administered dose reaches
the eye interior (Maurice DM and Mishima S, 1984). Systemic administration can be an
effective delivery method, but the high doses often needed to achieve effective intraocular
concentrations can cause systemic toxicity. For example, intravenous administration of
melphalan can be effective at treating retinoblastoma, but the doses required to achieve a
therapeutic response are associated with severe bone marrow toxicity (Shields and Shields,
2010). Periocular and intraocular administration are used to avoid poor drug penetration across
the ocular barriers. However, once inside the elimination rate of select drugs is rapid, often
necessitating the use of high doses, repeated administration or extended release systems to
counteract short drug half-lives (Geroski and Edelhauser, 2000;Yasukawa, et al., 2005). The
poor intraocular penetration of drugs administered outside of the eye and their rapid elimination
once inside suggests that active mechanisms contribute, along with passive processes (passive
diffusion across epithelia and aqueous outflow), to poor ocular drug delivery. Indeed, there is
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considerable evidence that drug transporters expressed in the blood-aqueous humor barrier,
blood-retinal barrier and cornea function to reduce the intraocular bioavailability of systemically
and topically administered drugs, and can facilitate their elimination from both the aqueous and
vitreous humor. Several previous reviews have discussed in detail drug transport by the bloodretinal barrier and cornea (Mannermaa, et al., 2006;Nakano, et al., 2014;Tomi and Hosoya,
2010;Chemuturi and Yanez, 2013;Jordan and Ruiz-Moreno, 2013;Hosoya and Tachikawa,
2009), whereas little attention has been given to the blood-aqueous humor barrier (i.e., iris-
Although there are numerous drug transporters that interact with organic electrolytes that
display a net negative charge (organic anions), a net positive charge (organic cations), or both
negative and positive charges (zwitterions) at physiological pH, most of our understanding of
drug transport by the iris-ciliary body come from studies using organic anions as ligands – i.e.,
substrates and/or inhibitors. Research in the 1960’s and 1970’s by Becker (Becker, 1960),
Forbes and Becker (Forbes and Becker, 1960), and Bárány (Barany, 1972;Barany,
1973b;Barany, 1973a;Barany, 1974;Barany, 1975;Barany, 1976) show that organic anion
transport by the iris-ciliary body has properties similar to those found in liver and kidney – the
two major excretory organs for drugs, and determinants of systemic pharmacokinetics. Even
back then, before the molecular identity of drug transporters were known, it was speculated that
these active transport mechanisms in the iris-ciliary body were important for ‘scavenging’ from
the aqueous and vitreous humor potential xenobiotic and endobiotic toxins that could be
deleterious to vision (Barany, 1976). This review covers 1) the tissue-, cellular and subcellular
expression of drug transporters in the iris-ciliary body, 2) in vitro evidence for drug transport
activity in the iris-ciliary body (with a focus on organic anions), and 3) in vivo evidence for the
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ciliary body).
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involvement of active transport in influencing intraocular drug concentrations, with a likely
contribution from the blood-aqueous humor barrier.
Drug transporters
Drug transporters support flux across plasma membranes of relatively small (<1000 mol
wt) organic molecules (organic anions, cations and zwitterions) that are structurally diverse.
Included within this class of chemicals are pharmacologically and toxicologically relevant
xenobiotics along with endogenous chemicals of physiological importance (endobiotics)
organic electrolytes across plasma membranes requires facilitated transport. Drug transporters
are expressed in a variety of barrier epithelia/endothelia, such as the intestinal epithelium, liver
hepatocytes, kidney tubules and brain capillary endothelium, where they influence tissue drug
concentrations, i.e., pharmacokinetics (Klaassen and Aleksunes, 2010). Drug transporters are
contained within two distinct families, the solute carrier (SLC) family and the ATP-binding
cassette (ABC) family. Transporters in the ABC family hydrolyze ATP to facilitate efflux of
their substrates out of cells. The multidrug resistance associated proteins (MRPs), breast cancer
resistant protein (BCRP) and P-glycoprotein (Pgp) are examples of drug transporters contained
within the ABC family. SLC drug transporters use a variety of energetic mechanisms to support
solute flux, including Na+-dependent co-transport, exchange and electrogenic facilitated
diffusion, and can facilitate cellular uptake or efflux depending on the electrochemical driving
forces. However, most of the SLC drug transporters discussed here support cellular
accumulation of their substrates in other tissues – the MATEs are an exception. Notable
examples of SLC drug transporters are the organic anion transporting polypeptides (OATPs),
organic anion transporters (OATs), organic cation transporters (OCTs) and multidrug and toxin
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(Klaassen and Aleksunes, 2010). Due to their charge at physiological pH, efficient movement of
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extrusion transporters (MATEs) – although there are others. A key feature of drug transporters is
their broad and sometimes overlapping ligand selectivity. SLC and ABC drug transporters likely
evolved with increasing physiological complexity to provide protection from exposure to an
increasing diversity of xenobiotics (Eraly, et al., 2004). Indeed, many of the SLC and ABC drug
transporters have arisen by gene duplication and display unique but also overlapping ligand
selectivity (Eraly, et al., 2004;Moitra and Dean, 2011). Within the ocular barriers drug
transporters likely function to protect tissues that are important for vision from exposure to
cornea. Importantly, many of the drug classes used to treat ocular disease contain small
molecule drugs that are substrates of drug transporters, such as antibiotics, antivirals, antiinflammatory drugs, α-adrenergic receptor agonists, prostaglandin analogs and antimetabolites.
The blood-aqueous humor barrier
The blood-aqueous humor barrier consists of the ciliary body epithelium and the tight
capillary endothelium of the iris (Cunha-Vaz, 1979). The ciliary body epithelium is responsible
for secretion of aqueous humor into the posterior chamber of the eye. The ciliary body extends
posteriorly from the iris root to the retina, forming a ring around the globe. The ciliary body has
two structurally distinct segments, the pars plicata and pars plana. The pars plicata is located
anteriorly and is characterized by extensive villi-like foldings called ciliary processes. The pars
plana is scalloped and is positioned between the pars plicata and the retina. The ciliary body is
highly vascularized by the choroidal capillaries, which are fenestrated and leaky (Friedenwald
and Stiehler, 1938;Stewart and Tuor, 1994). The iridial capillaries are continuous and contain
tight junctions, but have a higher permeability than inner retinal capillaries (i.e., the inner bloodretinal barrier) (Alm, 1992). The ciliary body is a bilayer comprised of two distinct cell layers –
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potentially toxic xenobiotics and endobiotics, especially the avascular lens epithelium and
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a pigmented cell layer and a non-pigmented cell layer. The pigmented and non-pigmented cells
are coupled via gap junctions (Shin, et al., 1996;Coffey, et al., 2002). The pigmented cells
contact the choroidal blood supply, whereas the basolateral membrane of the non-pigmented
epithelium contacts aqueous humor. Tight junctions are present in the non-pigmented epithelial
cells, but not in the pigmented cells (Sonsino, et al., 2002). Thus, the physical barrier to drug
movement across the ciliary body is the non-pigmented epithelium.
Expression of ABC transporters in iris-ciliary body
ABC drug transporters in isolated human iris-ciliary body (Zhang, et al., 2008;Chen, et al.,
2013), or in preparations containing only human iris or ciliary body that were isolated separately
by microdissection (Pelis, et al., 2009;Dahlin, et al., 2013;Lee, et al., 2015) – Table 1
summarizes the findings from these studies. In general, the studies are in good agreement with
each other. The majority of the studies observed mRNA for MRP1, MRP2, MRP3, MRP4,
MRP5, MRP6, MRP7, BCRP and Pgp in either ciliary body or iris-ciliary body preparations,
with only a few discrepancies. For example, Chen et al. (Chen, et al., 2013) failed to observe
mRNA for MRP3 in iris-ciliary body while Zhang et al. (Zhang, et al., 2008) did not detect
MRP2 mRNA in iris-ciliary body. Of the ABC transporter genes examined by Dahlin et al.
(Dahlin, et al., 2013), MRP5 and Pgp (MRP5>Pgp) showed the highest expression level in the
human ciliary body (Dahlin, et al., 2013). At the protein level (immunoblotting), MRP1, MRP2,
MRP3, MRP4, MRP6, MRP7 and Pgp were detected in human iris-ciliary body (Chen, et al.,
2013), and MRP1, MRP2, MRP4, BCRP and Pgp were detected in human ciliary body (Pelis, et
al., 2009) (Figure 1). While Pelis et al. (Pelis, et al., 2009) detected BCRP protein in human
ciliary body from a single donor by immunoblotting, another study failed to detect BCRP protein
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Several studies have examined mRNA (RT-PCR, qPCR or microarray) expression of
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expression in iris-ciliary body from multiple donors (Chen, et al., 2013). MRP2 localizes to the
apical membrane of non-pigmented epithelial cells of the human pars plana and plicata (Pelis, et
al., 2009), whereas MRP4 is located in the basolateral membrane of pigmented cells (Lee, et al.,
2015) (Figure 2). Both transporters are speculated to play a role in effluxing drugs from ciliary
epithelial cells into the choroid. In two independent studies, mRNA for MRP1, MRP3, MRP4,
MRP5, MRP6, MRP7 and Pgp were detected in human iris, whereas BCRP was low to
undetectable (Dahlin, et al., 2013;Lee, et al., 2015) (Table 1). Of the ABC transporter genes
most highly expressed. Although Dahlin et al (Dahlin, et al., 2013) detected MRP2 mRNA in
human iris, Lee et al. (Lee, et al., 2015) did not. Within the human iris, protein for MRP1,
MRP2, MRP4 and BCRP were observed (Pelis, et al., 2009) (Figure 1). Pgp has been localized
to iridial vessels by immunohistochemistry (Schlingemann, et al., 1998;Holash and Stewart,
1993) (Figure 2).
Expression of SLC transporters in the iris-ciliary body
The mRNA expression of numerous transporters in the SLC family have been examined
in the iris-ciliary body, ciliary body or iris of humans (Table 2). Most of the SLC transporters
listed are cellular uptake transporters. In some cases there is a discrepancy between the studies
regarding whether mRNA for the transporters are expressed in the ciliary body. Regardless,
from Table 2 it is apparent that both the ciliary body and iris express many of the SLC
transporters known to influence pharmacokinetics, and some transporters with important
physiological roles, such as transporters of bile acid acids (ASBT and NTCP), dicarboxylates
(NaDC3), peptides (PEPT1 and PEPT2), ergothioneine (OCTN1) and carnitine (OCTN2).
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examined in the iris by Dahlin et al. (Dahlin, et al., 2013), Pgp and MRP5 (Pgp>MRP5) were the
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Several of the transporters listed in Table 2 have been examined at the protein level in
human ciliary body by immunoblotting and/or immunohistochemistry. Protein for OATP1A2,
OATP1C1, OATP2B1, OATP3A1 and OATP4A1 were detected in human ciliary body extracts
by immunoblotting (Gao, et al., 2005) (Figure 1). Kraft et al. (Kraft, et al., 2010) also detected
mRNA for OATP2A1 and OATP2B1 by qPCR in human ciliary body as well as iris – albeit, the
expression of OATP2A1 in iris was near undetectable in their study. Gao et al. (Gao, et al.,
2005) demonstrated expression of OATP2B1, OATP3A1 and OATP4A1 protein in the
OATP1C1 were undetectable in the pars plicata (Figure 1). At odds with the study by Gao et al.
(Gao, et al., 2005), another study observed expression of OATP2B1 in basolateral membranes of
both non-pigmented cells as well as pigmented cells of the pars plicata (Kraft, et al., 2010)
(Figure 2). Thus, it is not clear based on this discrepancy whether OATP2B1 is present in the
basolateral membrane of pigmented cells of the pars plicata. OATP2A1 localizes to basolateral
membranes of pigmented cells and non-pigmented cells of the pars plicata (Kraft, et al., 2010)
(Figure 2). In the pars plana, OATP1A2 and OATP2B1 are restricted to the basolateral
membrane of non-pigmented epithelial cells, and OATP1C1, OATP3A1 and OATP4A1 are
present in basolateral membranes of non-pigmented epithelial cells as well as pigmented cells
(Gao, et al., 2005) (Figure 2). Both OATP2A1 and OATP2B1 also immunolocalize to iridial
vessels (Kraft, et al., 2010) (Figure 2). Importantly, prostaglandins are well-known substrates of
several OATPs (Hagenbuch and Stieger, 2013), and prostaglandin analogs, such as unoprostone
and latanoprost are anti-glaucoma drugs. The metabolite of unoprostone (unoprostone
carboxylate) is a substrate of OATP1A2, OATP2B1 and OATP4A1 (Gao, et al., 2005), and
latanoprost is a substrate of OATP2B1 and OATP2A1 (Kraft, et al., 2010), suggesting that
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basolateral membrane of non-pigmented epithelial cells of the pars plicata, while OATP1A2 and
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OATPs in the ciliary body may influence the pharmacological action of prostaglandin analogs
used to treat glaucoma.
In addition to several OATPs, protein for the renal organic anion transporters OAT1 and
OAT3 were detected in ciliary body from four different human donors by immunoblotting (Lee,
et al., 2015) (Figure 1). Within the kidney, both OAT1 and OAT3 use energy in the outwardlydirected α-ketoglutarate (Kreb’s cycle intermediate) gradient to facilitate cellular substrate
uptake via an anion exchange mechanism, and this gradient is maintained in part by the Na-
NaDC3 protein was detected in ciliary body from four human donors by immunoblotting (Lee, et
al., 2015) (Figure 1). Within the pars plicata of the human ciliary body both OAT1 and OAT3
are present in the basolateral membrane of non-pigmented epithelial cells, along with Na,KATPase (Figure 2) (Lee, et al., 2015). Although the subcellular localization of NaDC3 in ciliary
body is unknown, Nadc3 mRNA is present in non-pigmented epithelial cells of mice, as shown
by in situ hybridization (George, et al., 2004).
While the previous discussion of drug transporter expression in the blood-aqueous humor
barrier comes from studies that primarily used human tissues, in vitro and in vivo evidence
discussed below comes mostly from studies with other species (rabbit, monkey and bovine). It is
important to point out that species differences in drug transport exist. For example, whereas
humans have a single Pgp gene (ABCB1), rodents have two genes corresponding to Pgp
(Abcb1a and Abcb1b, also referred to as Mdr1a and Mdr1b). Regardless, many of the drug
transporter orthologs discussed in this review have ligand selectivities that are conserved across
species – example, Mdr1a and Mdr1b share many of the same ligands that interact with human
Pgp.
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dependent dicarboxylate cotransporter 3 (NaDC3) (Pelis and Wright, 2011). Accordingly,
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In vitro evidence for drug transporter activity in iris-ciliary body
In vitro data using fresh preparations of iris-ciliary body, or ciliary body devoid of iris
indicate that drug transporters, in particular, organic anion transporters, are active in the irisciliary body. The renocystographic agent iodohippurate and the cholecystographic agent
iodipamide, which actively accumulate in kidney tubules (Barany, 1972) and liver hepatocytes
(Joppen, et al., 1985), respectively, are actively taken up by the ciliary body (ciliary processes)
from a variety of species (Barany, 1972). Freshly excised segments of rabbit iris-ciliary body
is saturable, dependent on oxidative metabolism, temperature-sensitive, and inhibited by several
organic anions that are ligands of organic anion transporters, including para-aminohippurate,
chlorophenol red, penicillin and probenecid (Becker, 1960). Interestingly, iodopyracet is
actively secreted by renal tubules in a probenecid-sensitive manner, an active process likely
mediated by the classical renal organic anion secretory system, which involves the uptake
transporters OAT1 and/or OAT3 (Russel, et al., 1989). Chlorophenol red, which is secreted by
organic anion transporters in renal tubules, actively accumulates in rabbit ciliary body segments
that are devoid of iris (Sugiki, et al., 1961). That is, accumulation is metabolic- and temperaturedependent, and inhibited by iodopyracet, probenecid and penicillin (Sugiki, et al., 1961). Also,
the well-established substrate of the renal organic anion secretory system and OAT1 substrate
para-aminohippurate accumulates in the monkey ciliary body in vitro, and independently in the
iris as well (Stone, 1979). Para-aminohippurate accumulation by the monkey ciliary body and
iris is saturable, and active transport is inhibited by metabolic poisons, Na,K-ATPase inhibition
(ouabain), iodopyracet and probenecid (Stone, 1979). Several bile acids, including cholate,
glycocholate, deoxycholate and chenodeoxycholate show significant temperature-dependent
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accumulate iodopyracet to ~15-fold above the bath iodopyracet concentration, and accumulation
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accumulation by rabbit iris-ciliary body preparations, and accumulation is sensitive to
iodipamide (Barany, 1975). Consistent with these data, the ciliary body and iris express mRNA
for several bile acid uptake transporters, including ASBT, NTCP and several OATPs (Table 2).
Prostaglandin F2α accumulation by rabbit iris-ciliary body is saturable, and is inhibited by
metabolic poisons and a variety of organic anions, including probenecid, bromocresol green,
iodipamide and indomethacin, but is insensitive to the prototypic OAT1 substrate paraaminohippurate (Bito, et al., 1976). As indicated previously, prostaglandins are well-established
fresh preparations of iris-ciliary body show that the tissue(s) support active drug transport.
However, given the heterogeneity of cell types (hematopoietic, non-pigmented cells and
pigmented cells) expressed in this preparation, it is not possible from these studies to distinguish
the cellular source of the activity.
The aforementioned studies indicate that the iris-ciliary body expresses a variety of drug
transporters in the ABC and SLC families, and that the tissue can actively accumulate known
substrates of several SLC drug uptake transporters, but can the tissue support active
transepithelial transport of drugs? Two studies have in fact shown that the ciliary body mounted
in Ussing chambers can preferentially transport organic anions in the aqueous humor-to-blood
direction. The strength of the Ussing chamber technique is that by eliminating the chemical
(identical saline on both sides of the tissue) and electrical (voltage-clamped conditions) gradient
across the tissue preparation, it allows for the determination of ‘active’ transepithelial transport
of solutes. The organic anions tested were 6-carboxyfluorescein, para-aminohippurate and
estrone-3-sulfate, all of which are substrates of OAT1 and/or OAT3, and that are secreted by
renal proximal tubules (Pelis and Renfro, 2004). Kondo et al. (Kondo and Araie, 1994) showed
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substrates of several OATP uptake transporters (Hagenbuch and Stieger, 2013). The studies with
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a transport flux ratio (aqueous humor-to-blood flux/blood-to-aqueous humor flux) for 6carboxyfluorescein (10 μM) of ~5 across the rabbit ciliary body. The blood-to-aqueous humor
flux of 6-carboxyfluorescein occurs via passive diffusion, whereas the aqueous humor-to-blood
flux is saturable, with an apparent Michaelis constant of 28 μM (Kondo and Araie, 1994).
Active aqueous humor-to-blood flux of 6-carboxyfluorescein is inhibited by ouabain (Na+,K+ATPase inhibition), low bath [Na+], metabolic inhibition (2,4-dinitrophenol), low temperature,
probenecid, iodipamide and hippurate (Kondo and Araie, 1994). OAT1 and OAT3 are Na+-
dependent on the Na+-gradient for generation of the α-ketoglutarate gradient across the plasma
membrane, which in part is established by Na+,K+-ATPase and by Na+-dependent dicarboxylate
cotransporters. The Na+-dependence of active 6-carboxyfluorescein transport by the ciliary body
suggests coordination of Na+-dicarboxylate cotransport activity (likely NaDC3) and OATs in
active organic anion transport by the ciliary body epithelium. Consistent with the active
transport of 6-carboxyfluorescein in the aqueous humor-to-blood direction across the ciliary
body in Ussing chambers, the elimination rate of 6-carboxyfluorescein from the perfused bovine
eye in vitro is slowed ~5-fold by the OAT1 and OAT3 inhibitor, novobiocin, when it is coadministered to the aqueous humor (Lee, et al., 2015). The perfused bovine eye preparation
allows for administration of drugs directly into the aqueous humor, and for measurement of drug
appearance in the venous effluent due to the combined influence of passive aqueous humor
outflow and transport across the blood-aqueous humor barrier.
The flux ratio for para-aminohippurate transport (para-aminohippurate bath
concentration, 1 mM) and estrone-3-sulfate transport (estrone-3-sulfate bath concentration, 5
μM) across the bovine ciliary body in Ussing chambers was 5.3-fold and 3.5-fold, respectively –
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independent anion exchangers but cellular substrate uptake by the transporters is indirectly
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indicating preferential transport in the blood-to-aqueous humor direction (Lee, et al., 2015). Net
active transport of para-aminohippurate across the bovine ciliary body in the aqueous humor-toblood direction is inhibited by probenecid and novobiocin, but also by the MRP inhibitor MK571
(Lee, et al., 2015). The sensitivity of transepithelial organic anion transport to a variety of
inhibitors, the expression of OAT1 and OAT3 in the basolateral membrane of non-pigmented
epithelial cells, MRP2 in apical membrane of non-pigmented epithelial cells, and MRP4 in
basolateral membrane of pigmented cells (Figure 2), is in agreement with the active transport by
direction of elimination from the aqueous humor.
In vivo evidence for drug transporter function in the eye
In vivo studies using either systemic (intravenous or oral) or intraocular drug
administration provide evidence that drug transporters can reduce drug concentrations in the eye
either by limiting drug penetration across the ocular barriers (i.e., a functional barrier) and/or by
facilitating drug elimination once inside. The Pgp substrate cyclosporine A administered orally
to humans (BenEzra, et al., 1990), rabbits (BenEzra and Maftzir, 1990a) and rats (BenEzra and
Maftzir, 1990b) at therapeutic doses is not detected in the aqueous humor or vitreous, except
when the ocular barriers are disrupted by inflammation, suggesting that Pgp-mediated efflux
plays an important role in drug absorption across the ocular barriers following systemic
administration. Indeed, following intravenous administration of the Pgp substrates quinidine,
digoxin and verapamil, their aqueous humor uptake index values (concentration of drug in
aqueous humor/concentration of drug in blood) are lower than predicted based on their log D7.4
values, but are increased in the presence of Pgp inhibitors (Toda, et al., 2011)(Fujii, et al., 2014).
Kajikawa et al (Kajikawa, et al., 2000) showed that the aqueous humor clearance of the P-gp
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the ciliary body of para-aminohippurate, 6-carboxyfluorescein and estrone-3-sulfate in the
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substrate rhodamine-123 following its intracameral injection in rabbits is ~4-fold greater than
clearance due to passive aqueous humor outflow. Additionally, they found a 50% reduction in
the aqueous humor clearance of rhodamine-123 following co-administration of the Pgp inhibitor
quinidine (Kajikawa, et al., 2000). These results are similar to those of Senthikumari et al.
(Senthilkumari, et al., 2008), who showed following intravitreal injection a 2.6-fold increase in
vitreous rhodamine-123 levels with co-administration of the Pgp inhibitor, elacridar. Probably
the most definitive evidence for a role for Pgp in blood-aqueous humor barrier function in ocular
knockout (Pgp deficient) rats. Following intravenous administration in vivo of quinidine,
digoxin and verapamil, the aqueous humor uptake index values and the in vivo blood-to-tissue
influx permeability clearances of these Pgp substrates across the blood-aqueous humor barrier
were higher in Mdr1a knockout rats compared to wild-type rats (Fujii, et al., 2014).
In addition to Pgp substrates, several organic anions are actively eliminated from the eye
in vivo. The elimination of carbenecillin (Barza, et al., 1982), fluorescein and fluorescein
monoglucuronide (Kitano and Nagataki, 1986), and iodopyracet (Forbes and Becker, 1960) from
the rabbit eye, and cefazolin and carbenecillin from the monkey eye (Barza, et al., 1983)
following intravitreal administration are reduced following concomitant administration of
probenecid, a prototypical inhibitor of many drug transporters that interact with organic anions
(e.g., OATs, OATPs and MRPs). Furthermore, the elimination of iodopyracet from the rabbit
eye is a saturable process with a secretory maximum, consistent with the presence of active
transport (Becker and Forbes, 1961).
Although these in vivo data do not directly implicate a contribution of the iris-ciliary
body in limiting drug exposure in the eye, the expression of a variety of drug transporters in this
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drug disposition comes from a study done by Fujii et al (Fujii, et al., 2014) using Mdr1a
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tissue, and the presence of their activity in in vitro preparations, strongly suggest that the tissue
performs this function. It is important to note that the blood-retinal barrier and cornea also
express many drug transporters, and are most certainly important for ocular drug disposition.
The question remains, which blood ocular barrier is most important to ocular drug disposition?
The barrier most important is likely dependent on a variety of factors, including the site of drug
administration (topical, systemic, intravitreal or intracameral), the surface area of the tissues,
barriers to drug diffusion (e.g., unstirred fluid layers), and the abundance and activity of drug
Perspectives
The data presented indicate that the iris-ciliary body expresses a variety of drug
transporters that are likely involved in limiting drug exposure in the eye. However, it may be
possible to ‘high jack’ select drug transporters in order to enhance ocular drug bioavailability.
Indeed, drug transporters in other tissues have been targeted to improve the pharmacokinetic
profile of drugs. For example, PEPT1 has been targeted to increase the oral bioavailability of the
acyclovir pro-drug, valacyclovir (MacDougall and Guglielmo, 2004). Could PEPT1 in the irisciliary body be used to increase the ocular bioavailability of acyclovir for treatment of ocular
herpes virus infections? To take advantage of drug transporters in order to enhance ocular drug
bioavailability will require a better understanding of their cellular and subcellular distribution in
the iris-ciliary body, and the predominant direction in which they transport their drug substrates.
Although the focus of this review is on drug transporters and their influence on ocular
pharmacokinetics, many of the transporters present in the iris-ciliary body play important
physiological roles, yet we lack an understanding of their importance in ocular physiology. For
example, what purpose do ergothioneine (OCTN1), carnitine (OCTN2), peptide (PEPT1 and
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transporters in these tissues.
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PEPT2) and cyclic nucleotide (MRP4 and MRP5) transporters serve in the iris-ciliary body?
Ergothioneine is a naturally occurring amino acid derivative that has antioxidant properties.
Although it is not made in the human body, some human tissues contain high concentrations of
ergothioneine. Relatively high levels of ergothioneine are present in bovine and porcine ocular
tissues, including aqueous humor, where it likely protects ocular tissues from oxidative damage
(Shires, et al., 1997). Carnitine is important for conversion of fatty acids into metabolic energy,
and as an antioxidant. Within the eye of animals levels of -carnitine are highest in the iris,
potentially slow age-related ocular pathologies, such as age-related macular degeneration
(Pescosolido, et al., 2008). Future work should examine the influence of OCTN1 and OCTN2
on levels of ergothioneine and carnitine in ocular tissues. Aqueous humor contains a variety of
components that absorb ultraviolet radiation (UV), including ascorbate, uric acid, proteins and
the amino acids tyrosine and tryptophan (Ringvold, et al., 2003). Perhaps peptide transporters in
the iris-ciliary body contribute to the UV-absorbing potential of aqueous humor. The cAMPadenosine signaling pathway modulates aqueous humor inflow (Farahbakhsh, 2003). The ability
of MRP4 and MRP5 to modulate intracellular and extracellular cAMP levels may make these
transporters novel targets for glaucoma therapy. A better understanding of the role drug
transporters in the blood-aqueous humor barrier play in ocular pharmacokinetics and physiology
should lead to improved treatment of ocular diseases.
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ciliary body, and choroid-retina, and the use of -carnitine and its derivatives in humans may
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Authorship Contributions
Wrote or contributed to the writing of the manuscript: JL and RMP
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19
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References
Alm A (1992) Ocular Circulation, in Adler's Physiology of the Eye (Hart WJ ed) pp 198-227,
Mosby-Year Book, Inc, St. Louis.
Barany EH (1972) Inhibition by hippurate and probenecid of in vitro uptake of iodipamide and
o-iodohippurate. A composite uptake system for iodipamide in choroid plexus, kidney cortex and
anterior uvea of several species. Acta Physiol Scand 86:12-27.
Barany EH (1973a) The liver-like anion transport system in rabbit kidney, uvea and choroid
plexus. I. Selectivity of some inhibitors, direction of transport, possible physiological substrates.
Acta Physiol Scand 88:412-429.
Barany EH (1974) Bile acids as inhibitors of the liver-like anion transport system in the rabbit
kidney, uvea and choroid plexus. Acta Physiol Scand 92:195-203.
Barany EH (1975) In vitro uptake of bile acids by choroid plexus, kidney cortex and anterior
uvea. I. The iodipamide-sensitive transport systems in the rabbit. Acta Physiol Scand 93:250268.
Barany EH (1976) Organic cation uptake in vitro by the rabbit iris-ciliary body, renal cortex, and
choroid plexus. Invest Ophthalmol 15:341-348.
Barar J, Javadzadeh AR and Omidi Y (2008) Ocular novel drug delivery: impacts of membranes
and barriers. Expert Opin Drug Deliv 5:567-581.
Barza M, Kane A and Baum J (1982) The effects of infection and probenecid on the transport of
carbenicillin from the rabbit vitreous humor. Invest Ophthalmol Vis Sci 22:720-726.
Barza M, Kane A and Baum J (1983) Pharmacokinetics of intravitreal carbenicillin, cefazolin,
and gentamicin in rhesus monkeys. Invest Ophthalmol Vis Sci 24:1602-1606.
Becker B (1960) The transport of organic anions by the rabbit eye. I. In vitro iodopyracet
(Diodrast) accumulation by ciliary body-iris preparations. Am J Ophthalmol 50:862-7.:862-867.
Becker B and Forbes M (1961) Iodopyracet (diodrast) transport by the rabbit eye. Am J Physiol
200:461-464.
BenEzra D and Maftzir G (1990a) Ocular penetration of cyclosporin A. The rabbit eye. Invest
Ophthalmol Vis Sci 31:1362-1366.
BenEzra D and Maftzir G (1990b) Ocular penetration of cyclosporine A in the rat eye. Arch
Ophthalmol 108:584-587.
20
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 4, 2017
Barany EH (1973b) The liver-like anion transport system in rabbit kidney, uvea and choroid
plexus. II. Efficiency of acidic drugs and other anions as inhibitors. Acta Physiol Scand 88:491504.
DMD Fast Forward. Published on February 19, 2016 as DOI: 10.1124/dmd.116.069369
This article has not been copyedited and formatted. The final version may differ from this version.
DMD # 69369
BenEzra D, Maftzir G, de Court and Timonen P (1990) Ocular penetration of cyclosporin A. III:
The human eye. Br J Ophthalmol 74:350-352.
Bito LZ, Davson H and Salvador EV (1976) Inhibition of in vitro concentrative prostaglandin
accumulation by prostaglandins, prostaglandin analogues and by some inhibitors of organic
anion transport. J Physiol 256:257-271.
Chemuturi N and Yanez JA (2013) The role of xenobiotic transporters in ophthalmic drug
delivery. J Pharm Pharm Sci 16:683-707.
Chen P, Chen H, Zang X, Chen M, Jiang H, Han S and Wu X (2013) Expression of efflux
transporters in human ocular tissues. Drug Metab Dispos 41:1934-1948.
Cunha-Vaz J (1979) The blood-ocular barriers. Surv Ophthalmol 23:279-296.
Dahlin A, Geier E, Stocker SL, Cropp CD, Grigorenko E, Bloomer M, Siegenthaler J, Xu L,
Basile AS, Tang-Liu DD and Giacomini KM (2013) Gene expression profiling of transporters in
the solute carrier and ATP-binding cassette superfamilies in human eye substructures. Mol
Pharm 10:650-663.
Eraly SA, Monte JC and Nigam SK (2004) Novel slc22 transporter homologs in fly, worm, and
human clarify the phylogeny of organic anion and cation transporters. Physiol Genomics.
Farahbakhsh NA (2003) Purinergic signaling in the rabbit ciliary body epithelium. J Exp Zool A
Comp Exp Biol 300:14-24.
Forbes M and Becker B (1960) The transport of organic anions by the rabbit eye. II. In vivo
transport of iodopyracet (Diodrast). Am J Ophthalmol 50:867-875.
Friedenwald JS and Stielher RD (1938) Circulation of the Aqueous: VII. A mechanism of
secretion of the intraocular fluid. Arch Ophthal 20:761-786.
Fujii S, Setoguchi C, Kawazu K and Hosoya K (2014) Impact of P-glycoprotein on blood-retinal
barrier permeability: comparison of blood-aqueous humor and blood-brain barrier using mdr1a
knockout rats. Invest Ophthalmol Vis Sci 55:4650-4658.
Gao B, Huber RD, Wenzel A, Vavricka SR, Ismair MG, Reme C and Meier PJ (2005)
Localization of organic anion transporting polypeptides in the rat and human ciliary body
epithelium. Exp Eye Res 80:61-72.
Gaudana R, Ananthula HK, Parenky A and Mitra AK (2010) Ocular drug delivery. AAPS J
12:348-360.
21
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 4, 2017
Coffey KL, Krushinsky A, Green CR and Donaldson PJ (2002) Molecular profiling and cellular
localization of connexin isoforms in the rat ciliary epithelium. Exp Eye Res 75:9-21.
DMD Fast Forward. Published on February 19, 2016 as DOI: 10.1124/dmd.116.069369
This article has not been copyedited and formatted. The final version may differ from this version.
DMD # 69369
George RL, Huang W, Naggar HA, Smith SB and Ganapathy V (2004) Transport of Nacetylaspartate via murine sodium/dicarboxylate cotransporter NaDC3 and expression of this
transporter and aspartoacylase II in ocular tissues in mouse. Biochim Biophys Acta 1690:63-69.
Geroski DH and Edelhauser HF (2000) Drug delivery for posterior segment eye disease. Invest
Ophthalmol Vis Sci 41:961-964.
Hagenbuch B and Stieger B (2013) The SLCO (former SLC21) superfamily of transporters. Mol
Aspects Med 34:396-412.
Holash JA and Stewart PA (1993) The relationship of astrocyte-like cells to the vessels that
contribute to the blood-ocular barriers. Brain Res 629:218-224.
Joppen C, Petzinger E and Frimmer M (1985) Properties of iodipamide uptake by isolated rat
hepatocytes. Naunyn Schmiedebergs Arch Pharmacol 331:393-397.
Jordan J and Ruiz-Moreno JM (2013) Advances in the understanding of retinal drug disposition
and the role of blood-ocular barrier transporters. Expert Opin Drug Metab Toxicol 9:1181-1192.
Kajikawa T, Mishima HK, Murakami T and Takano M (2000) Role of P-glycoprotein in ocular
clearance of rhodamine 123 in rabbits. Pharm Res 17:479-481.
Kitano S and Nagataki S (1986) Transport of fluorescein monoglucuronide out of the vitreous.
Invest Ophthalmol Vis Sci 27:998-1001.
Klaassen CD and Aleksunes LM (2010) Xenobiotic, bile acid, and cholesterol transporters:
function and regulation. Pharmacol Rev 62:1-96.
Kondo M and Araie M (1994) Movement of carboxyfluorescein across the isolated rabbit irisciliary body. Curr Eye Res 13:251-255.
Kraft ME, Glaeser H, Mandery K, Konig J, Auge D, Fromm MF, Schlotzer-Schrehardt U,
Welge-Lussen U, Kruse FE and Zolk O (2010) The prostaglandin transporter OATP2A1 is
expressed in human ocular tissues and transports the antiglaucoma prostanoid latanoprost. Invest
Ophthalmol Vis Sci 51:2504-2511.
Lee J, Shahidullah M, Hotchkiss A, Coca-Prados M, Delamere NA and Pelis RM (2015) A
renal-like organic anion transport system in the ciliary epithelium of the bovine and human eye.
Mol Pharmacol 87:697-705.
MacDougall C and Guglielmo BJ (2004) Pharmacokinetics of valaciclovir. J Antimicrob
Chemother 53:899-901.
22
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 4, 2017
Hosoya K and Tachikawa M (2009) Inner blood-retinal barrier transporters: role of retinal drug
delivery. Pharm Res 26:2055-2065.
DMD Fast Forward. Published on February 19, 2016 as DOI: 10.1124/dmd.116.069369
This article has not been copyedited and formatted. The final version may differ from this version.
DMD # 69369
Mannermaa E, Vellonen KS and Urtti A (2006) Drug transport in corneal epithelium and bloodretina barrier: emerging role of transporters in ocular pharmacokinetics. Adv Drug Deliv Rev
58:1136-1163.
Maurice DM and Mishima S (1984) Ocular pharmacokinetics, in Handbook of Pharmacology pp
19-116, Springer-Verlag, Berline.
Moitra K and Dean M (2011) Evolution of ABC transporters by gene duplication and their role
in human disease. Biol Chem 392:29-37.
Nakano M, Lockhart CM, Kelly EJ and Rettie AE (2014) Ocular cytochrome P450s and
transporters: roles in disease and endobiotic and xenobiotic disposition. Drug Metab Rev 46:247260.
Pelis RM and Renfro JL (2004) Role of tubular secretion and carbonic anhydrase in vertebrate
renal sulfate excretion. Am J Physiol Regul Integr Comp Physiol 287:R491-R501.
Pelis RM and Wright SH (2011) Renal transport of organic anions and cations, in
Comprehensive Physiology pp 1795-1835, John Wiley & Sons, Inc..
Pescosolido N, Imperatrice B and Karavitis P (2008) The aging eye and the role of L-carnitine
and its derivatives. Drugs R D 9:3-14.
Ringvold A, Anderssen E, Jellum E, Bjerkas E, Sonerud GA, Haaland PJ, Devor TP and
Kjonniksen I (2003) UV-Absorbing compounds in the aqueous humor from aquatic mammals
and various non-mammalian vertebrates. Ophthalmic Res 35:208-216.
Russel FG, Wouterse AC and Van Ginneken CA (1989) Physiologically based pharmacokinetic
model for the renal clearance of iodopyracet and the interaction with probenecid in the dog.
Biopharm Drug Dispos 10:137-152.
Schlingemann RO, Hofman P, Klooster J, Blaauwgeers HGT, Van Der Gaag R and Vrensen GF
(1998) Ciliary muscle capillaries have blood-tissue barrier characteristics. Exp Eye Res 66, 747754.
Senthilkumari S, Velpandian T, Biswas NR, Saxena R and Ghose S (2008) Evaluation of the
modulation of P-glycoprotein (P-gp) on the intraocular disposition of its substrate in rabbits.
Curr Eye Res 33:333-343.
Shields CL and Shields JA (2010) Intra-arterial chemotherapy for retinoblastoma: the beginning
of a long journey. Clin Experiment Ophthalmol 38:638-643.
Shin BC, Suzuki T, Tanaka S, Kuraoka A, Shibata Y and Takata K (1996) Connexin 43 and the
glucose transporter, GLUT1, in the ciliary body of the rat. Histochem Cell Biol 106:209-214.
23
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 4, 2017
Pelis RM, Shahidullah M, Ghosh S, Coca-Prados M, Wright SH and Delamere NA (2009)
Localization of multidrug resistance-associated protein 2 in the nonpigmented ciliary epithelium
of the eye. J Pharmacol Exp Ther 329:479-485.
DMD Fast Forward. Published on February 19, 2016 as DOI: 10.1124/dmd.116.069369
This article has not been copyedited and formatted. The final version may differ from this version.
DMD # 69369
Shires TK, Brummel MC, Pulido JS and Stegink LD (1997) Ergothioneine distribution in bovine
and porcine ocular tissues. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 117:117120.
Sonsino J, Gong H, Wu P and Freddo TF (2002) Co-localization of junction-associated proteins
of the human blood--aqueous barrier: occludin, ZO-1 and F-actin. Exp Eye Res 74:123-129.
Stewart PA and Tuor UI (1994) Blood-eye barriers in the rat: correlation of ultrastructure with
function. J Comp Neurol 340:566-576.
Stone RA (1979) The transport of para-aminohippuric acid by the ciliary body and by the iris of
the primate eye. Invest Ophthalmol Vis Sci 18:807-818.
Toda R, Kawazu K, Oyabu M, Miyazaki T and Kiuchi Y (2011) Comparison of drug
permeabilities across the blood-retinal barrier, blood-aqueous humor barrier, and blood-brain
barrier. J Pharm Sci 100:3904-3911.
Tomi M and Hosoya K (2010) The role of blood-ocular barrier transporters in retinal drug
disposition: an overview. Expert Opin Drug Metab Toxicol 6:1111-1124.
Yasukawa T, Ogura Y, Sakurai E, Tabata Y and Kimura H (2005) Intraocular sustained drug
delivery using implantable polymeric devices. Adv Drug Deliv Rev 57:2033-2046.
Zhang T, Xiang CD, Gale D, Carreiro S, Wu EY and Zhang EY (2008) Drug transporter and
cytochrome P450 mRNA expression in human ocular barriers: implications for ocular drug
disposition. Drug Metab Dispos 36:1300-1307.
24
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 4, 2017
Sugiki S, Constant MA and Becker B (1961) In vitro accumulation of chlorphenol red by rabbit
ciliary body. J Cell Comp Physiol 58:181-183.
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Figure Legends
Figure 1. Schematic diagram of the anterior globe highlighting ABC and SLC drug transporters
shown to be expressed in dissections of human iris-ciliary body, ciliary body or iris by
immunoblotting. See text for references to the published studies.
Figure 2. Cellular and subcellular expression of protein for ABC and SLC drug transporters in
the human ciliary body (left panel) and iris (right panel) determined by immunohistochemistry.
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*, indicates discrepancies between studies (see text). BLM, basolateral membrane; NPE, nonpigmented epithelium; PC, pigmented cell. See text for references to the published studies.
25
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Table 1. Expression of mRNA for ABC drug transporters in iris-ciliary body (ICB), ciliary body
(CB) or iris (I) from human.
Transporter
Zhang
ICB
+
+
Dahlin
CB
+
+
+
+
+
+
Lee
CB
Dahlin
I
+
+
+
+
+
+
Lee
I
MRP1
MRP2
+
MRP3
+
+
MRP4
+
+
MRP5
+
+
MRP6
+/+
MRP7
+
+
+
BCRP
+
+
+
+/+/P-gp
+
+
+
+
+
+
The studies by Chen et al. (Chen, et al., 2013), Dahlin et al. (Dahlin et al., 2013), Lee et al. (Lee
et al., 2015), Pelis et al. (Pelis et al., 2009) and Zhang et al. (Zhang et al., 2008) used qPCR,
microarray, microarray, RT-PCR and qPCR for mRNA detection, respectively. -, not detected.
+, detected. +/-, indicates that mRNA expression was low to negligible. Blank areas indicate
that the study did not examine expression of the respective ABC drug transporter. The number
of individual donor eyes used in these studies are as follows: Chen et al. (n=6), Dahlin et al.
(n=15), Lee et al. (n=5-6), Pelis et al. (n=1) and Zhang et al. (n=3).
26
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Chen
ICB
+
+
+
+
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Table 2. Expression of mRNA for SLC drug transporters in iris-ciliary body (ICB), ciliary body
(CB) or iris (I) from human.
Transporter
Zhang
ICB
+
Dahlin
CB
+
+
+
+
Dahlin
I
+
+
+/+
Lee
I
+/+
+/+/+
+
+/+
+
+
OATP1A2
OATP1B1
OATP1B3
OATP2B1
OATP1C1
Organic anion
OATP2A1
transporting
OATP3A1
polypeptides
OATP4A1
OATP4C1
OATP5A1
OATP6A1
OAT1
+
+
+
OAT2
+
+
Organic anion
OAT3
+
+
+
transporters
OAT4
ASBT
+
+
Bile acid
NTCP
+
+
transporters
NaDC1
+/Dicarboxylate
transporters
NaDC3
+
+
OCT1
+
+
+
OCT2
+
+
+
OCT3
+
+
+
Organic cation
OCTN1
+
transporters
OCTN2
+
+
+
MATE1
+
+
MATE2
+
+
PEPT1
+/+
+
Peptide
PEPT2
+
transporters
The studies by Zhang et al. (Zhang et al., 2008), Dahlin et al. (Dahlin et al., 2013) and Lee et al.
(Lee et al., 2015) used qPCR, microarray and microarray for mRNA detection, respectively. -,
not detected. +, detected. +/-, indicates that mRNA expression was low to negligible. Blank areas
indicate that the study did not examine expression of the respective SLC drug transporter. The
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Lee
CB
+/+
+/+
+
+
+/+
+
+/-
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number of individual donor eyes used in these studies are as follows: Dahlin et al. (n=15), Lee et
al. (n=5-6) and Zhang et al. (n=3).
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28
aqueous
humor
ciliary
body
iris
MRP1, MRP2,
MRP4, BCRP
leens
iris-ciliary body
pars plicata
pars plana
vitreous
hum
mor
Retinal pigmented
epithelium
: tight enddothelial cells
: fenestraated endothelial cells
epithelium
: retinal pigmented
p
: ciliary eppithelium (bilayer)
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MRP1, MRP2, MRP4,
Pgp, BCRP, OATP1A2,
OATP1C1, OATP2B1,
OATP3A1, OATP4A1,
OAT1, OAT3, NaDC3
corrnea
MRP1, MRP2,
MRP3, MRP4,
MRP6, MRP7,
Pgp
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igure 1
Fig
CILIARY BODY
pigmented cell
tight
junction
nonpigmented cell
IRIS
MRP4
(BLM, PC)
BLM, PC
pars plana
OATP2A1
OATP2B1*
OATP1C1
OATP3A1
OATP4A1
MRP2 (apical, NPE
PE)
tight junction
OAT1
OAT3
OATP2B1
OATP2A1
OATP3A1
OATP4A1
BLM, NPE
pars plicata
OATP1A2
OATP1C1
OATP2B1
OATP3A1
OATP4A1
BLM, NPE
pars plana
iridial
capillary
OATP2A1
OATP2B1
Pgp
aqueous humo
mor
choroid
ciliary epithelium
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BLM, PC
pars plicata
gap
junction
Subcellular
localization unknown
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gure 2
Figur