Outward-Directed Transport





Introduction


Several barriers protect the eye from exogenous compounds that may exert toxic reactions in the eye. These barriers can be classified anatomically into three groups. First, the eye is protected from the air and lacrimal fluid by the tight epithelial barriers of the cornea and conjunctiva. Second, the tight tissue barriers in the iris and ciliary body form the blood-aqueous barrier (BAB) between the blood circulation and aqueous humor. Third, blood-retinal barriers (BRBs) (inner barrier: retinal capillaries; outer barrier: retinal pigment epithelium [RPE]) regulate molecular transfer between the neural retina and systemic blood circulation. The entire barrier function of these tissues includes several key components: (1) a physical barrier formed by tight intercellular junctions, (2) the metabolism of xenobiotics, and (3) transport activity (inward and outward transport).


In this chapter, ocular outward transport is discussed in the context of pharmacology, toxicology, and pathophysiology. Outward transport shuttles compounds from the eye to the lacrimal fluid and systemic blood circulation. This action is accomplished mostly by efflux transporters that actively transport compounds from the intracellular space to the extracellular environment. In the context of outward transport from the eye, those efflux transporters located at the barriers between the eye and lacrimal fluid or eye and blood circulation are the most interesting ones.


The most anterior ocular barrier consists of the epithelia of the cornea and conjunctiva that line the lacrimal fluid ( Fig. 17.1 ). Outward transport in these tissues protects the eye from compounds in the lacrimal fluid. In the case of drug administration, the outward transport must compete with inward passive diffusion and inward active transport, thereby leading to relative changes, but not necessarily complete block of drug transport from the lacrimal fluid into the eye. The final outcome of such competition depends on the physicochemical properties of the drug (affecting passive diffusion and transport) and the expression of relevant transporters in the cornea and conjunctiva. Thus, ocular absorption of topically applied drugs depends on the barrier properties of the corneal and conjunctival epithelia and expression of transporters. It should be noted that conjunctival permeability of small molecules is about nine times higher than their permeation in the cornea. Overall, transcorneal bioavailability of topically applied small-molecule drugs in the aqueous humor is approximately 0.1% to 5%, whereas biologics have negligible absorption to the cornea. Ocular bioavailability across conjunctiva has not been quantitated, but the transconjunctival route tends to be more important for hydrophilic compounds with poor corneal permeability.




Fig. 17.1


Tissue barriers of the eye. BAB , Blood-aqueous barrier; BRB , blood-retinal barrier; RPE , retinal pigment epithelium.


The BRB regulates molecular transfer between the posterior eye segment and the systemic circulation. The BRB is composed of retinal vascular endothelium (inner BRB) and retinal-pigmented epithelium (outer BRB). The inner BRB is composed of a tight monolayer of endothelial cells, and the outer BRB is composed of retinal pigmented epithelial cells that form a monolayer with tight junctions. Bruch’s membrane and the choroid are relatively leaky tissues providing a negligible barrier compared with the RPE. In general, the BRB allows the permeation of small molecules with adequate lipophilicity, but restricts serious permeation of proteins and protein-bound drugs in the plasma. , Likewise, the BRB limits the elimination of large molecules from the vitreous, prolonging the vitreal half-lives of injected biologics, such as vascular endothelial growth factor inhibitors, for up to several days. On the contrary, small molecules, capable of permeating the BRB, are rapidly eliminated from the vitreous with half-lives of a few hours. Outward transport in the BRB may limit retinal access of systemically circulating compounds, whereas it may accelerate elimination of vitreal compounds from the eye, thereby reducing retinal exposure to the exogenous substances.


This chapter comprises a discussion on the efflux transporters on the ocular surface (cornea, conjunctiva) and the BRB (retinal capillaries, RPE). Information about the biologic and pharmacologic roles of efflux transporters in the eye is still emerging and changing. This chapter provides an update of current knowledge about the expression and functionality of the efflux transporters in the eye. The main focus will be on outward transport in the cornea and BRB, as they have been investigated more than the conjunctiva and BAB.


Efflux transporters


The efflux transporters are transmembrane proteins that are expressed in the liver and kidney, where they contribute to the pharmacokinetics of various drugs and their metabolites. In physiologic barriers such as the intestine, blood-brain barrier (BBB), and placenta, the efflux transporters protect the body from harmful xenobiotics and excrete endogenous metabolites. Additionally, cancer cells often overexpress efflux transporters to provide protection from anticancer agents. In fact, P-glycoprotein (P-gp) was initially found through its ability to provide drug resistance in colchicine-selected Chinese hamster ovary cells. P-gp was later called multidrug resistance protein 1 (MDR1), based on its involvement in the resistance toward a broad range of anticancer drugs such as paclitaxel and doxorubicin. Breast cancer resistance protein (BCRP) and the multidrug resistance–associated proteins (MRPs) were also initially found through their involvement in multidrug resistance in cancer cells, which is reflected in their persisting unofficial nomenclature. The efflux transporters belong to the ATP-binding cassette (ABC) family of transporters, which in humans contains 48 members, divided into 7 subfamilies, A–G. Not all the transporters are involved in drug transport; however, the most important ones involved in drug transport are listed in Table 17.1 . In the eye, efflux transporters have been found at several barrier tissues—for example, the cornea and BRB.



Table 17.1

Efflux transporters with pharmacokinetic impact












































Gene name Protein name Uniprot ID Example drug substrates
ABCB1 P-gp, MDR1 P08183 Erythromycin, methotrexate, ciprofloxacin
ABCC1 MRP1 P33527 Methotrexate
ABCC2 MRP2 Q92887 Erythromycin, methotrexate
ABCC3 MRP3 O15438 Methotrexate
ABCC4 MRP4 O15439 Methotrexate, ganciclovir
ABCC5 MRP5 O15440 Acyclovir, latanoprost
ABCG2 BCRP Q9UNQ0 Ciprofloxacin, methotrexate

ABC , ATP-binding cassette; BCRP , Breast cancer resistance protein; MDR1 , multidrug resistance protein 1; MRP , multidrug resistance–associated protein; P-gp , P-glycoprotein.


The ABC transporters share the same structure, consisting of two transmembrane domains and two cytoplasmic adenosine triphosphate (ATP) -binding domains ( Table 17.2 ; Fig. 17.2A ). The energy from ATP is used to drive the transport of substrates over the membrane against the concentration gradient (i.e., from lower concentration toward higher concentration). The transmembrane domain containing the substrate cavity consists of 12 transmembrane helices. Some efflux transporters contain five additional transmembrane helices forming an N-terminal transmembrane domain, with a largely unknown function ( Fig. 17.2B ). Other ABC transporters are so-called half-transporters, where the peptide chain only forms one ATP-binding domain and six transmembrane helices. In the ABCG family, the order of the ATP-binding domain and the transmembrane helices is reversed ( Fig. 17.2C ). Two of these half-transporters need to dimerize to form a functional unit.



Table 17.2

ABC transporter family and members identified in human ocular tissues







































Family Function Members Ocular tissue
ABCA Cholesterol and lipid transport ABCA4 Retina
ABCB Transport peptides, toxins etc. P-glycoprotein BRB, BAB
ABCC Mainly ion transport


  • MRP1



  • MRP2



  • MRP3



  • MRP4



  • MRP5




  • Cornea, BAB, BRB



  • Cornea, BAB



  • Cornea



  • Cornea, BAB, BRB



  • Cornea, BRB

ABCD Peroxisomal transport
ABCE/ABCF No transport function, only ATP-binding domain
ABCG Transport ions, lipids, toxins, etc. BCRP


  • Cornea, BAB



  • Retina


ABC , ATP-binding cassette; BAB , blood-aqueous barrier; BCRP , breast cancer resistance protein; BRB , blood-retinal barrier.



Fig. 17.2


Schematic structures of ATP-binding cassette (ABC) transporters. ( A ) Protein with 12 transmembrane helices and 2 cytoplasmic adenosine triphosphate (ATP)–binding sites. ( B ) Protein with five additional transmembrane helices. ( C ) “Reverse” half-transporters with six transmembrane helices and single ATP-binding site. The N- and C- terminus of the peptide chain is indicated with an amino group (NH2) and a carboxyl group (COOH), respectively.


Methods of studying transporters


Transporters can be investigated in terms of structure, substrate specificity, expression, localization, and functionality. Most data on transporter structure, substrates, and general functionality are available in the general biologic and medical literature. In this section, we briefly summarize some methods that are relevant for understanding the expression and functions of efflux proteins in outward-directed transport in the eye.


Proteomic analysis


Historically, expression of proteins in the eye has been widely studied with western blots, but modern mass spectrometry–based methods of proteomics can provide much more complete information on protein expression, including in the eye. As transporter proteins regulate substrate movement across the plasma membrane of the cells, identification and quantification of the proteins facilitates understanding of ocular physiology and pharmacology. Global proteomics, comprehensive untargeted protein identification using mass spectrometry, has been used to reveal transporter and relevant protein profiles in the RPE cells, whereas quantitative targeted proteomics, a precise protein quantification, has been used to determine the amounts of transporter proteins in the RPE using liquid chromatography tandem mass spectrometry. As transmembrane domains of transporter proteins are water insoluble, peptides with specific amino acid sequences of transporter protein are usually selected from water-soluble extracellular domains. Efflux transporter protein expression may vary under different conditions, such as oxidative stress and inflammation. A combination of phospho-proteomic analysis of phosphorylated proteins and quantitative targeted proteomics is also a useful methodology, providing fundamental insights into the cellular mechanisms of the functional changes of efflux transporters. , Overall, proteomic techniques are useful for generating data on transporter expression in the ocular barriers, as exemplified in the case of RPE cells. However, such data are not available yet for the cornea, conjunctiva, or BAB.


Overexpressing cells


The function of efflux transporters is often studied at a cellular level in overexpression systems because this approach enables the study of a specific transporter in a controlled environment. Dissecting the specific role of a single efflux transporter type can be difficult because substrate specificities of efflux transporters are relatively wide and often overlap. The pharmacologic impact of the transporter in overexpressed cell systems cannot be reliably assessed because the transporter amount does not necessarily correspond to the in vivo situation and many other transporters with overlapping substrate specificity may contribute in vivo. The intact cells may not be appropriate for efflux transporter studies if the substrate has poor transcellular permeability or the cell does not express relevant uptake transporters because the compound usually needs to enter the cells before binding to the efflux transporters. Instead, ATP-dependent substrate transport by individual efflux transporters can be studied in inverted cell membrane vesicles. Assays measuring only the ATPase activity of the efflux transporters are not ideal, as the transporters have a basal ATPase activity, and not all substrates or inhibitors significantly change ATP consumption. Detection of the accumulated substrate in the vesicles is more accurate.


Ocular cell and tissue models


Expression and function of efflux transporters have been investigated in cell lines and primary cells. It is important to note that cell lines may differ significantly from their in vivo counterparts, thus leading to potentially misleading conclusions. For example, the widely used human corneal epithelial (HCE) cell line overexpressed several efflux transporters at mRNA compared with normal human corneal epithelial cells that expressed only BCRP, MRP1, and MRP5. Moreover, transcriptomic analysis revealed major differences in the protein expression patterns of differentiated HCE cell line and normal human cornea. In contrast, the widely used human RPE cell line ARPE-19 showed similar expression of efflux transporters as primary human RPE cells, but another cell line (D407) showed a very different expression pattern. Overall, cell model and cell culture conditions must be carefully selected to avoid misleading conclusions about efflux transport of compounds in vivo. ,


Isolated tissues such as the cornea and RPE are often used to characterize molecular transport in the eye. The rabbit is the most commonly used species in ocular pharmacokinetics and the most relevant species for in vitro to in vivo translation, but the differences or similarity in transporter protein expression between rabbit and human is not known. Porcine and bovine tissues have also been used in permeation studies in vitro. Ussing chambers enable the study of directionality, which can be used to assess the impact of efflux transport on overall net transfer of compounds in the barrier. An example is a study with bovine RPE-choroid, which shows the importance of maintaining and controlling tissue viability. Data on protein expression and transport in vitro can be further extended toward in vivo predictions by using pharmacokinetic models. , In vivo translation is not straightforward because there are several confounding factors such as contribution of passive diffusion, differences in expression levels and protein sequence, and possible tissue alterations during in vitro experiments. However, increasing experimental data and improving computational capacity enhance the value of such predictions.


In vivo animal experiments


Kinetic ocular in vivo studies are performed with rabbits. In general, the rabbit is considered a relevant model that provides a reasonable human translation of pharmacokinetics in terms of topical and intravitreal drug administration. However, the validity of the rabbit model for efflux transporter functions is still unclear. It is difficult to dissect the functions of efflux transport from the overall pharmacokinetics that are affected by various factors. Some approaches from BBB studies could be applicable also in the ocular context. These approaches include prediction of K p,uu,brain (i.e., the steady state unbound drug concentration ratio between blood and the brain). This is based on the pharmacokinetic model, with an efflux transporter expression level per surface area of BBB (µmol/cm 2 ), an intrinsic efflux rate of drug per transporter protein (µL/[min × µmol of transporter protein]), and the passive diffusion rate of drug per surface area of BBB (µL/[min × cm 2 ]).


Corneal outward transport


The cornea serves as a barrier to harmful substances and drug molecules owing to the tight multilayered epithelium, but also because of the presence of efflux transporters. Fig. 17.3 illustrates the efflux transporters that are known to be expressed in the human and rabbit cornea. The protein expression levels have been determined in primary cells or tissues using western blot or immunohistochemical staining, but absolute and relative protein expression data are lacking still.




Fig. 17.3


Efflux transport protein expressed in the cornea and conjunctiva. Purple: expressed only in rabbit; pink: expressed both in rabbit and human; turquoise: expressed only in human. , , , , , BCRP , Breast cancer resistance protein; MRP , multidrug resistance–associated protein; P-gp , P-glycoprotein.


The impact of corneal efflux transporters in drug delivery has been studied mainly in rabbits, using either excised cornea or in vivo models. The P-gp substrate, rhodamine 123, had a more than twofold larger basolateral-to-apical permeability in an isolated rabbit cornea compared with the opposite apical-to-basolateral permeability. Additionally, the directionality was inhibited by the P-gp inhibitor, verapamil. Comparable results were seen in rabbit in vivo using a single-dose infusion method, in which a small well was placed on top of the cornea containing the P-gp substrate, erythromycin. The bioavailability of erythromycin in the anterior chamber was increased by up to fourfold in the presence of P-gp inhibitors.


Erythromycin was shown also to be a substrate of the MRP2 efflux transporter. , The aqueous humor bioavailability of erythromycin increased by 2.5- to 4-fold in the presence of P-gp and MRP inhibitors, using the single-dose infusion method in rabbits. The involvement of MRP2 in erythromycin transport was also studied in excised rabbit cornea. The efflux protein P-gp was first inhibited by verapamil and then the remaining MRP2 efflux transport was inhibited by MK571, resulting in a similar permeability of erythromycin in both directions.


Functionally active MRP1 and MRP5 efflux transporters were shown in an uptake assay with isolated rabbit cornea. Concentration differences were seen for 5(6)-carboxy-2′,7′-dichlorofluorescein (CDCF) substrate in the presence of an MRP1 and MRP5 inhibitor, probenecid. CDCF is a polar substrate produced intracellularly by hydrolysis of CDCF diacetate (CDCFDA), and it cannot exit the cell without efflux transporters. Probenecid, an inhibitor of MRP2, affected efflux of CDCF from the cells. , The presence of MRP2 and MRP5 activity in the rabbit cornea was supported by an in vivo study in which the bioavailability of acyclovir was increased 2.2-fold in the presence of the MRP2 and MRP5 inhibitor, MK571.


MRP3 expression was observed in the rabbit cornea, but no activity was detected for MRP3 and BCRP after the use of methotrexate as a substrate. MRP4 activity was not detected in the rabbit cornea, using adefovir in the presence of an indomethacin inhibitor. However, in the human cornea, a variant of MRP4 caused lower intraocular pressure after latanoprost treatment.


Species-dependent expression of efflux transporters is important to consider when trying to translate functionality studies into clinical settings ( Fig. 17.3 ). For example, the function of P-gp has been studied in rabbits, but the transporter is not expressed in the human cornea. , , Conversely, the clinically relevant efflux transporters, MRP4 and BCRP, are present in the human cornea but not in the rabbit. , , ,


The role of corneal efflux transporters in topical drug delivery is most likely modest due to the high drug concentrations used clinically. However, it was predicted that corneal efflux transporters have a high impact on the aqueous humor bioavailability of drugs at applied concentrations of 0.1% to 1% if the drug has low passive permeability and high efflux transporter affinity. Drugs released from pharmaceutical suspensions and controlled release devices might be more prone to efflux transport, owing to lower drug concentrations in the tear fluid and inside the corneal epithelial cells; however, many other factors may influence the overall clinical impact of efflux transporters.


Blood-retinal barrier outward transport


The BRB consists of retinal capillary endothelium that forms the inner BRB and the RPE that forms the outer BRB ( Fig. 17.4 ). The outer BRB has an important role in outward transport from the eye as permeation across the RPE is the main elimination route for low-molecular-weight drugs from the vitreous. Both inner and outer BRB contain several efflux proteins that may contribute to their barrier function and outward transport from the eye.


Jun 29, 2024 | Posted by in OPHTHALMOLOGY | Comments Off on Outward-Directed Transport

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