Expression, activity and pharmacokinetic impact of ocular transporters.

The eye is protected by several tissues that limit the permeability and entry of potentially harmful substances, but also hamper the delivery of drugs in the treatment of ocular diseases. Active transport across the ocular barriers may affect drug distribution, but the impact of drug transporters on ocular drug delivery is not well known. We have collected and critically reviewed the literature for ocular expression and activity of known drug transporters. The review concentrates on drug transporters that have been functionally characterized in ocular tissues or primary cells and on transporters for which there is available expression data at the protein level. Species differences are highlighted, since these may explain observed inconsistencies in the influence of specific transporters on drug disposition. There is variable evidence about the pharmacokinetic role of transporters in ocular tissues. The strongest evidence for the role of active transport is available for the blood-retinal barrier. We explored the role of active transport in the cornea and blood retinal barrier with pharmacokinetic simulations. The simulations show that the active transport is important only in the case of specific parameter combinations.

Pharmacokinetics, Efficacy, and Safety of the Preservative-free Fixed Combination of Tafluprost 0.0015% and Timolol 0.5% in Healthy Volunteers: A Phase I Comparison vs. the Corresponding Preservative-free Monotherapies.

PURPOSE: Plasma concentrations of tafluprost acid and timolol were compared after single (Day 1) and repeated (Day 8) instillations of once-daily tafluprost 0.0015%-timolol 0.5% preservative-free (PF) fixed-dose combination (FDC), once-daily PF tafluprost 0.0015%, and twice-daily PF timolol 0.5%. PATIENTS AND METHODS: Fifteen healthy volunteers were randomized to this double-masked, single-center, three-period cross-over study. A wash-out interval of at least 4 weeks separated each three 8-day dosing period. Blood samples were drawn on the first and last day of each dosing period, prior to the morning dose, as well as 5, 10, 15, 30, and 45 min, and 1, 1.5, 2, 4, 8, and 12 h post-dosing. Sample plasma concentrations of tafluprost acid and/or timolol were determined and maximum concentration (C max), area under the concentration-over-time curve from time zero to the last time point with a quantifiable measurement (AUC0-last), and time to maximum concentration were calculated. Intraocular pressure (IOP), adverse events, and ocular/systemic safety variables were also evaluated. RESULTS: Plasma concentrations of tafluprost acid were low, with similar levels measured subsequent to either single or repeated dosing of PF FDC and PF tafluprost. On both sampling days, concentrations peaked at 10 min after the dose, and were cleared from the blood circulation by 30 min; average C max ranged from 17 to 24 pg/mL, and AUC0-last from 3 to 5 pg*h/mL. Plasma concentrations of timolol were comparable after the first dose of PF FDC or PF timolol. Concentrations peaked at 15 min post-dose and diminished in a similar manner after 2 h; average C max was 800 pg/mL and AUC0-last 3900 pg*h/mL. As expected, PF timolol produced a higher Day 8 pre-dose timolol concentration than PF FDC (235 vs. 37 pg/mL; p < 0.001, respectively). The Day 8 post-dose changes in timolol concentrations were relative to this pre-dose difference. All study treatments were well tolerated and safe. PF FDC seemed to provide the best IOP reduction. CONCLUSIONS: PF FDC demonstrated good IOP-lowering efficacy and displayed similar pharmacokinetic characteristics to the monotherapy agents. Exposure to timolol was reduced via the halved dosing.

A critical assessment of in vitro tissue models for ADME and drug delivery.

Cultured cells are widely used in the evaluation of new drugs and drug delivery systems. Cells can be grown at different levels of complexity ranging from simple reductionist models to complex organotypic models. The models are based on primary, secondary or stem cell derived cell cultures. Generation of tissue mimics with cultured cells is a difficult task, because the tissues have well-defined morphology, complex protein expression patterns and multiple inter-linked functions. Development of organotypic cell culture models requires proper biomaterial matrix and cell culture protocols that are able to guide the cells to the correct phenotype. This review illustrates the critical features of the cell culture models and, then, selected models are discussed in more detail (epidermal, corneal epithelial, retinal pigment epithelium, and hepatocyte models). The cell models are critically evaluated paying attention to the level of characterization and reliability of in vivo translation. Properties of the cell models must be characterized in detail using multiple biological assays and broad sets of model drugs. Robust in vivo predictions can be achieved with well-characterized cell models that are used in combination with computational methods that will bridge the gap between in vitro cell experiments and physiological situation in vivo in the body.

Regulation of the human tyrosinase gene in retinal pigment epithelium cells: the significance of transcription factor orthodenticle homeobox 2 and its polymorphic binding site.

PURPOSE: Tyrosinase is the rate-limiting enzyme responsible for melanin biosynthesis in the retinal pigment epithelium (RPE) of the eye. Melanin has an important role in retinal development, function, and protection against light-induced oxidative stress, and melanin levels are associated with age-related macular degeneration (AMD). Because the levels of and protection afforded by melanin seem to decline with increasing age, proper regulation of the human tyrosinase gene (TYR) in the RPE is an important but insufficiently understood process. Our purpose was to obtain detailed information on regulation of the TYR gene promoter in the human RPE and to specify the role of orthodenticle homeobox 2 (OTX2) and microphthalmia-associated transcription factor (MITF). METHODS: We used luciferase reporter constructs to study regulation of the human TYR gene promoter in cultured human RPE cells. We further studied the role of OTX2 and MITF, their binding sites, and endogenous expression by using mutagenesis, electrophoretic mobility shift assay, yeast two-hybrid assay, RNA interference, and gene expression analyses. RESULTS: In the RPE, OTX2 activated the human TYR gene promoter via direct trans-activation of novel OTX2 binding elements. In addition, we found that indirect activation by OTX2 via more proximal MITF binding sites, even in the absence of OTX2 sites, took place. These results are consistent with the physical interaction observed between OTX2 and MITF. Overexpression or knockdown of OTX2 in RPE cells resulted in corresponding changes in tyrosinase mRNA expression. Finally, we found that a single nucleotide polymorphism (SNP rs4547091) at the most proximal OTX2 binding site is associated with altered nuclear protein binding and a remarkable decrease in TYR promoter activity in RPE cells. This single nucleotide polymorphism (SNP) is more common in the European population in which AMD is also more prevalent. CONCLUSIONS: In the RPE, OTX2 activates the human TYR gene promoter by direct DNA binding and by interaction with MITF. Such synergistic interaction highlights the role of OTX2 as a potential coregulator of numerous MITF target genes in the eye. Genetic differences in OTX2 binding sites affect tyrosinase regulation. Collectively, these findings emphasize the role of OTX2 in regulating the human TYR gene, with implications for inter-individual differences in melanin synthesis, retinal development, and function as well as susceptibility to retinal degeneration associated with aging.

Efflux protein expression in human stem cell-derived retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells in the back of the eye nourish photoreceptor cells and form a selective barrier that influences drug transport from the blood to the photoreceptor cells. At the molecular level, ATP-dependent efflux transporters have a major role in drug delivery in human RPE. In this study, we assessed the relative expression of several ATP-dependent efflux transporter genes (MRP1, -2, -3, -4, -5, -6, p-gp, and BCRP), the protein expression and localization of MRP1, MRP4, and MRP5, and the functionality of MRP1 efflux pumps at different maturation stages of undifferentiated human embryonic stem cells (hESC) and RPE derived from the hESC (hESC-RPE). Our findings revealed that the gene expression of ATP-dependent efflux transporters MRP1, -3, -4, -5, and p-gp fluctuated during hESC-RPE maturation from undifferentiated hESC to fusiform, epithelioid, and finally to cobblestone hESC-RPE. Epithelioid hESC-RPE had the highest expression of MRP1, -3, -4, and P-gp, whereas the most mature cobblestone hESC-RPE had the highest expression of MRP5 and MRP6. These findings indicate that a similar efflux protein profile is shared between hESC-RPE and the human RPE cell line, ARPE-19, and suggest that hESC-RPE cells are suitable in vitro RPE models for drug transport studies. Embryonic stem cell model might provide a novel tool to study retinal cell differentiation, mechanisms of RPE-derived diseases, drug testing and targeted drug therapy.

Barrier analysis of periocular drug delivery to the posterior segment.

Periocular administration is a potential way of delivering drugs to their targets in posterior eye segment (vitreous, neural retina, retinal pigment epithelium (RPE), choroid). Purpose of this study was to evaluate the role of the barriers in periocular drug delivery. Permeation of FITC-dextrans and oligonucleotides in the bovine sclera was assessed with and without Pluronic gel in the donor compartment. Computational model for subconjunctival drug delivery to the choroid and neural retina/vitreous was built based on clearance concept. Kinetic parameters for small hydrophilic and lipophilic drug molecules, and a macromolecule were obtained from published ex vivo and in vivo animal experiments. High negative charge field of oligonucleotides slows down their permeation in the sclera. Pluronic does not provide adequate rate control to modify posterior segment drug delivery. Theoretical calculations for subconjunctival drug administration indicated that local clearance by the blood flow and lymphatics removes most of the drug dose which is in accordance with experimental results. Calculations suggested that choroidal blood flow removes most of the drug that has reached the choroid, but this requires experimental verification. Calculations at steady state using the same subconconjunctival input rate showed that the choroidal and vitreal concentrations of the macromolecule is 2-3 orders of magnitude higher than that of small molecules. The evaluation of the roles of the barriers augments to design new drug delivery strategies for posterior segment of the eye.

Filter-cultured ARPE-19 cells as outer blood-retinal barrier model.

Retinal pigment epithelium (RPE) regulates drug transfer between posterior eye segment and blood circulation, but there is no established RPE cell model for drug delivery studies. We evaluated ARPE-19 filter culture model for this purpose. Passive permeability of 6-carboxyfluorescein, betaxolol and FITC-dextran (40kDa) and active transport of 6-carboxyfluorescein, sodium fluorescein, rhodamine 123, cyclosporine A and digoxin in ARPE-19 model were investigated and compared with isolated bovine RPE-choroid tissue. In addition, barrier properties, and mRNA expression of RPE-specific and melanogenesis-related genes (RPE65, VMD2, CRALBP, OTX-2, MITF-A, TRP-1, tyrosinase) were measured in various culture conditions. The filter grown ARPE-19 cell model showed reasonable barrier properties (TER close to 100Omegacm(2)), but its permeability was slightly higher than that of isolated bovine RPE/choroid specimens. In active transport studies the ARPE-19 model mimics qualitatively the permeability profile of bovine RPE-choroid, but ARPE-19 model underestimates the importance of active transport relative to passive diffusion. Long-term filter-cultured ARPE-19 cells expressed various RPE-specific and melanogenesis-related genes at higher levels than the ARPE-19 cells cultured short-term in flasks. ARPE-19 model can be used to study drug permeation processes in the RPE.

Effluxing ABC transporters in human corneal epithelium.

ATP-binding cassette (ABC) transporters are able to efflux their substrate drugs from the cells. We compared expression of efflux proteins in normal human corneal epithelial tissue, primary human corneal epithelial cells (HCEpiC), and corneal epithelial cell culture model (HCE model) based on human immortal cell line. Expression of multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1-6 (MRP1-6) and breast cancer resistance protein (BCRP) was studied using quantitative RT-PCR, Western blot, and immunohistochemistry. Only MRP1, MRP5, and BCRP were expressed in the freshly excised human corneal epithelial tissue. Expression of MRP1 and MRP5 was localized predominantly in the basal cells of the central cornea and limbus. Functional efflux activity was shown in the cell models, but they showed over-expression of most efflux transporters compared to that of normal corneal epithelium. In conclusion, MRP1, MRP5, and BCRP are expressed in the corneal epithelium, but MDR1, MRP2, MRP3, MRP4, and MRP6 are not significantly expressed. HCE cell model and commercially available primary cells deviate from this expression profile.

Efflux protein expression in human retinal pigment epithelium cell lines.

PURPOSE: The objective of this study was to characterize efflux proteins (P-glycoprotein (P-gp), multidrug resistance proteins (MRP1-6) and breast cancer resistance protein (BCRP)) of retinal pigment epithelium (RPE) cell lines. METHODS: Expression of efflux proteins in two secondary (ARPE-19, D407) and two primary (HRPEpiC and bovine) RPE cell lines was measured by quantitative RT-PCR and western blotting. Furthermore, activity of MRP1 and MRP5 of ARPE-19 cell line was assessed with calcein-AM and carboxydichlorofluorescein (CDCF) probes. RESULTS: Similar efflux protein profile was shared between ARPE-19 and primary RPE cells, whereas D407 cell line was notably different. D407 cells expressed MRP2 and BCRP, which were absent in other cell lines and furthermore higher MRP3 transcript expression was found. MRP1, MRP4 and MRP5 were identified from all human RPE cell lines and MRP6 was not expressed in any cell lines. The pattern of efflux protein expression did not change when ARPE-19 cells were differentiated on filters. The calcein-AM and CDCF efflux tests provided evidence supporting MRP1 and MRP5 activity in ARPE-19 cells. CONCLUSIONS: MRP1, MRP4 and MRP5 are the main efflux transporters in RPE cell lines. There are differences in efflux protein expression between RPE cell lines.

Crosstalk between Hsp70 molecular chaperone, lysosomes and proteasomes in autophagy-mediated proteolysis in human retinal pigment epithelial cells.

The pathogenesis of age-related macular degeneration involves chronic oxidative stress, impaired degradation of membranous discs shed from photoreceptor outer segments and accumulation of lysosomal lipofuscin in retinal pigment epithelial (RPE) cells. It has been estimated that a major part of cellular proteolysis occurs in proteasomes, but the importance of proteasomes and the other proteolytic pathways including autophagy in RPE cells is poorly understood. Prior to proteolysis, heat shock proteins (Hsps), agents that function as molecular chaperones, attempt to refold misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates. In the present study, the roles of the Hsp70 molecular chaperone and proteasomal and lysosomal proteolytic pathways were evaluated in human RPE cells (ARPE-19). The Hsp70 and ubiquitin protein levels and localization were analysed by Western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate the morphological changes. Hsp70 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay. The proteasome inhibitor MG-132 evoked the accumulation of perinuclear aggregates positive for Hsp70, ubiquitin-protein conjugates and the lysosomal membrane protein LAMP-2. Interestingly, the hsp70 mRNA depletion significantly increased cell death in conjunction with proteasome inhibition. We found that the accumulation of lysosomes was reversible: a cessation of proteasome inhibition led to clearance of the deposits via a mechanism believed to include autophagy. The molecular chaperone Hsp70, proteasomes and autophagy have an important regulatory role in the protein turnover of human RPE cells and may thus open new avenues for understanding degenerative processes in retinal cells.

Radicicol but not geldanamycin evokes oxidative stress response and efflux protein inhibition in ARPE-19 human retinal pigment epithelial cells.

Drug delivery to retinal cells has represented a major challenge for ophthalmologists for many decades. However, drug targeting to the retina is essential in therapies against retinal diseases such as age-related macular degeneration, the most common reason of blindness in the developed countries. Retinal cells are chronically exposed to oxidative stress that contributes to cellular senescence and may cause neovascularization in the most severe age-related macular degeneration cases. Various pre- and clinical studies have revealed that heat shock protein 90 (HSP90) inhibitors, such as geldanamycin and radicicol, are promising drugs in the treatment of different malignant processes. In this study, our goal was to compare the effects of 0.1 microM, 1 microM or 5 microM geldanamycin or radicicol on the oxidative stress response, cytotoxicity, and efflux protein activity (a protein pump which removes drugs from cells) in ARPE-19 (human retinal pigment epithelial, RPE) cells. Our findings indicate that geldanamycin and radicicol increased HSP70 and HSP27 expression analyzed by western blotting. Cellular levels of protein carbonyls were increased in response to 0.1 microM (P=0.048 for 24 h, P=0.018 for 48 h) or 5 microM (P=0.030 for 24 h, P=0.046 for 48 h) radicicol but not to geldanamycin analyzed by ELISA assay. In addition, HNE-protein adducts were accumulated in the RPE cells exposed to 0.1 microM or 5 microM radicicol but not to geldanamycin analyzed by western blotting. However, MTT assay revealed that 5 microM geldanamycin reduced cellular viability 20-30% (P<0.05 for 24 h, P<0.01 for 48 h), but this was not observed at any radicicol concentration in RPE cells. Interestingly, the increased oxidative stress response was associated with efflux protein inhibition (20-30%) when the cells were exposed to 1 microM or 5 microM (P<0.05) radicicol, but not in geldanamycin-treated RPE cells. These novel findings help in understanding the influence of HSP90 inhibition and regulatory mechanisms of drug delivery to retinal cells.

The effect of 17beta-estradiol on IL-6 secretion and NF-kappaB DNA-binding activity in human retinal pigment epithelial cells.

Toll-like receptors (TLRs) and inflammatory cascades participate in the pathology of age-related macular degeneration (AMD). The effect of estrogens on the development of AMD is poorly understood, although many studies indicate that these compounds can modulate inflammatory responses. In this study, we investigated the regulatory role of TLR agonists and 17beta-estradiol (E(2)) on IL-6 expression and NF-kappaB DNA-binding activity in human retinal pigment epithelial cells (ARPE-19). The inflammatory response of ARPE-19 cells to various TLR agonists, e.g. Pam, zymosan, flagellin, SLTA and lipopolysaccharide (LPS) exposures were examined via the secretion of IL-6 cytokine as analyzed by ELISA. In addition, the IL-6 responses to the estrogen-receptor agonist, E(2), and to the estrogen-receptor antagonist ICI 182.780 as well as to the NF-kappaB inhibitor helenalin were compared. The DNA-binding activity of NF-kappaB transcription factor of nuclear cell extracts was analyzed by the gel mobility shift assay (EMSA). TLR4 gene expression was studied by quantitave PCR. The TLR4 agonist, LPS, caused a clear IL-6 response that was attenuated by E(2) in ARPE-19-cells. The anti-inflammatory properties of E(2) were mediated through estrogen receptors and were associated with decreased NF-kappaB DNA-binding activity. The level of TLR4 gene expression was not affected by LPS exposure. Our results indicate that IL-6 expression is regulated through NF-kappaB transcription factor and stereoid-receptor signalling pathways in ARPE-19 cells.

Drug transport in corneal epithelium and blood-retina barrier: emerging role of transporters in ocular pharmacokinetics.

Corneal epithelium and blood-retina barrier (i.e. retinal capillaries and retinal pigment epithelium (RPE)) are the key membranes that regulate the access of xenobiotics into the ocular tissues. Corneal epithelium limits drug absorption from the lacrimal fluid into the anterior chamber after eyedrop administration, whereas blood-retina barrier restricts the entry of drugs from systemic circulation to the posterior eye segment. Like in general pharmacokinetics, the role of transporters has been considered to be quite limited as compared to the passive diffusion of drugs across the membranes. As the functional role of transporters is being revealed it has become evident that the transporters are widely important in pharmacokinetics. This review updates the current knowledge about the transporters in the corneal epithelium and blood-retina barrier and demonstrates that the information is far from complete. We also show that quite many ocular drugs are known to interact with transporters, but the studies about the expression and function of those transporters in the eye are still sparse. Therefore, the transporters probably have greater role in ocular pharmacokinetics than we currently realise.

Long-lasting secretion of transgene product from differentiated and filter-grown retinal pigment epithelial cells after nonviral gene transfer.

PURPOSE: The purpose of this study was to investigate the extent, duration, and direction of transgene expression after nonviral gene transfer to differentiated retinal pigment epithelial (RPE) cells. METHODS: Polarized human RPE cells (ARPE-19) were transfected with nonviral vectors [DOTAP/DOPE with and without protamine sulfate (PS), DOTAP, PEI (polyethyleneimine), DHP-12] using secreted alkaline phosphatase (SEAP) as a reporter gene. Cellular uptake was studied by flow cytometry. RESULTS: Up to 80-fold differences were observed in the peak reporter gene expression. The highest peak levels and the longest lifetime of SEAP expression (> 69 days) were obtained with DOTAP/DOPE/PS/pDNA complexes. With PEI, higher expression was seen to the apical side than to the basolateral side. CONCLUSIONS: In contrast to most differentiated epithelial cells, the differentiated RPE cells can be transfected at high and prolonged levels with selected lipoplexes.