Aldehyde oxidase 1 activity and protein expression in human, rabbit, and pig ocular tissues.

Aldehyde oxidase (AOX) is a cytosolic drug-metabolizing enzyme which has attracted increasing attention in drug development due to its high hepatic expression, broad substrate profile and species differences. In contrast, there is limited information on the presence and activity of AOX in extrahepatic tissues including ocular tissues. Because several ocular drugs are potential substrates for AOX, we performed a comprehensive analysis of the AOX1 expression and activity profile in seven ocular tissues from humans, rabbits, and pigs. AOX activities were determined using optimized assays for the established human AOX1 probe substrates 4-dimethylamino-cinnamaldehyde (DMAC) and phthalazine. Inhibition studies were undertaken in conjunctival and retinal homogenates using well-established human AOX1 inhibitors menadione and chlorpromazine. AOX1 protein contents were quantitated with targeted proteomics and confirmed by immunoblotting. Overall, DMAC oxidation rates varied over 10-fold between species (human ˃˃ rabbit ˃ pig) and showed 2- to 6-fold differences between tissues from the same species. Menadione seemed a more potent inhibitor of DMAC oxidation across species than chlorpromazine. Human AOX1 protein levels were highest in the conjunctiva, followed by most posterior tissues, whereas anterior tissues showed low levels. The rabbit AOX1 expression was high in the conjunctiva, retinal pigment epithelial (RPE), and choroid while lower in the anterior tissues. Quantification of pig AOX1 was not successful but immunoblotting confirmed the presence of AOX1 in all species. DMAC oxidation rates and AOX1 contents correlated quite well in humans and rabbits. This study provides, for the first time, insights into the ocular expression and activity of AOX1 among multiple species.

Selective drug delivery to the retinal cells: Biological barriers and avenues.

Retinal drug delivery is a challenging, but important task, because most retinal diseases are still without any proper therapy. Drug delivery to the retina is hampered by the anatomical and physiological barriers resulting in minimal bioavailability after topical ocular and systemic administrations. Intravitreal injections are current method-of-choice in retinal delivery, but these injections show short duration of action for small molecules and low target bioavailability for many protein, gene based drugs and nanomedicines. State-of-art delivery systems are based on prolonged retention, controlled drug release and physical features (e.g. size and charge). However, drug delivery to the retina is not cell-specific and these approaches do not facilitate intracellular delivery of modern biological drugs (e.g. intracellular proteins, RNA based medicines, gene editing). In this focused review we highlight biological factors and mechanisms that form the basis for the selective retinal drug delivery systems in the future. Therefore, we are presenting current knowledge related to retinal membrane transporters, receptors and targeting ligands in relation to nanomedicines, conjugates, extracellular vesicles, and melanin binding. These issues are discussed in the light of retinal structure and cell types as well as future prospects in the field. Unlike in some other fields of targeted drug delivery (e.g. cancer research), selective delivery technologies have been rarely studied, even though cell targeted delivery may be even more feasible after local administration into the eye.

Intravitreal CendR peptides target laser-induced choroidal neovascularization sites in mice.

Choroidal neovascularization (CNV) is a common ocular pathology that may be associated in a variety of eye diseases. Although intravitreal injection treatment of anti-vascular endothelial growth factor (anti-VEGF) drugs shows significant clinical benefits in CNV treatment, the limitations of the current therapy need to be addressed. The aim of our study was to investigate the potential utility of three C-end Rule (CendR) peptides (RPARPAR, PL3, iRGD) for CNV targeting and to evaluate the efficacy of peptides for treating experimental CNV in mice. We observed that the CendR peptides localize to the CNV lesion sites after intravitreal injection and were mainly found in the outer nuclear cell layer (ONL) of the mouse retina. Interestingly, experimental therapy with tenascin-C (TNC-C) and neuropilin-1 (NRP-1)-targeting PL3 peptide, reduced angiogenesis and decreased vascular leakage. The results suggest that PL3 and potentially other CendR peptides could serve as affinity targeting ligands and therapeutics for ocular diseases that involve pathological CNV.

Structural Features Affecting the Interactions and Transportability of LAT1-Targeted Phenylalanine Drug Conjugates.

L-type amino acid transporter 1 (LAT1) transfers essential amino acids across cell membranes. Owing to its predominant expression in the blood-brain barrier and tumor cells, LAT1 has been exploited for drug delivery and targeting to the central nervous system (CNS) and various cancers. Although the interactions of amino acids and their mimicking compounds with LAT1 have been extensively investigated, the specific structural features for an optimal drug scaffold have not yet been determined. Here, we evaluated a series of LAT1-targeted drug-phenylalanine conjugates (ligands) by determining their uptake rates by in vitro studies and investigating their interaction with LAT1 via induced-fit docking. Combining the experimental and computational data, we concluded that although LAT1 can accommodate various types of structures, smaller compounds are preferred. As the ligand size increased, its flexibility became more crucial in determining the compound's transportability and interactions. Compounds with linear or planar structures exhibited reduced uptake; those with rigid lipophilic structures lacked interactions and likely utilized other transport mechanisms for cellular entry. Introducing polar groups between aromatic structures enhanced interactions. Interestingly, compounds with a carbamate bond in the aromatic ring's para-position displayed very good transport efficiencies for the larger compounds. Compared to the ester bond, the corresponding amide bond had superior hydrogen bond acceptor properties and increased interactions. A reverse amide bond was less favorable than a direct amide bond for interactions with LAT1. The present information can be applied broadly to design appropriate CNS or antineoplastic drug candidates with a prodrug strategy and to discover novel LAT1 inhibitors used either as direct or adjuvant cancer therapy.

Hydroquinone predisposes for retinal pigment epithelial (RPE) cell degeneration in inflammatory conditions.

In addition to hypoxia, inflammation is capable of inducing vascular endothelial growth factor (VEGF) expression in human retinal pigment epithelial (RPE) cells. Excessive levels of VEGF promote choroidal neovascularization and thereby contribute to the pathogenesis of wet age-related macular degeneration (AMD). Intravitreal anti-VEGF injections ameliorate pathological vessel neoformation in wet AMD but excessive dampening of VEGF can result in a degeneration of the RPE. In the present study, we induced VEGF production by exposing human ARPE-19 cells to the pro-inflammatory IL-1α and subsequently to hydroquinone, a component of tobacco smoke that is a major environmental risk factor for AMD. Effects were monitored by measuring the levels of VEGF and anti-angiogenic pigment epithelium-derived factor (PEDF) using an enzyme-linked immunosorbent assay (ELISA) technique. In addition, we measured the production of reactive oxygen species (ROS) using the 2',7'-dichlorofluorescin diacetate (H2DCFDA) probe and studied the effects of two anti-oxidants, ammonium pyrrolidinedithiocarbamate (APDC) and N-acetyl-cysteine (NAC), on VEGF production. Cellular and secreted VEGF as well as secreted PEDF levels were reduced at all tested hydroquinone concentrations (10, 50, or 200 µM); these effects were evident prior to any reduction of cell viability evoked by hydroquinone. Cell viability was carefully explored in our previous study and verified by microscoping in the present study. APDC further reduced the VEGF levels, whereas NAC increased them. The 50 µM concentration of hydroquinone increased ROS production in ARPE-19 cells primed with IL-1α. Hydroquinone disturbs the regulatory balance of VEGF and PEDF in inflammatory conditions. These data support the idea that hydroquinone mediates RPE degeneration by reducing VEGF levels and may predispose to dry AMD since VEGF is as well important for retinal integrity.

Mechanisms of cellular retention of melanin bound drugs: Experiments and computational modeling.

Melanin binding of drugs is known to increase drug concentrations and retention in pigmented eye tissues. Even though the correlation between melanin binding in vitro and exposure to pigmented eye in vivo has been shown, there is a discrepancy between rapid drug release from melanin particles in vitro and the long in vivo retention in the pigmented tissues. We investigated mechanisms and kinetics of pigment-related drug retention experimentally using isolated melanin particles from porcine retinal pigment epithelium and choroid, isolated porcine eye melanosomes, and re-pigmented ARPE-19 cells in a dynamic flow system. The experimental studies were supplemented with kinetic simulations. Affinity and capacity of levofloxacin, terazosin, papaverine, and timolol binding to melanin revealed Kd values of ≈ 50-150 µM and Bmax ≈ 40-112 nmol.mg-1. The drugs were released from melanin in <1 h (timolol) or in 6-12 h (other drugs). The drugs were released slower from the melanosomes than from melanin; the experimental differences ranged from 1.2-fold (papaverine) to 7.4-fold (timolol). Kinetic simulations supported the role of the melanosomal membrane in slowing down the release of melanin binders. In release studies from the pigmented ARPE-19 cells, drugs were released from the cellular melanin to the extracellular space in ≈ 1 day (timolol) and ≈ 11 days (levofloxacin), i.e., much slower than the release from melanin or melanosomes. Simulations of drug release from pigmented cells in the flow system matched the experimental data and enabled further sensitivity analyses. The simulations demonstrated a significant prolongation of drug retention in the cells as a function of decreasing drug permeability in the melanosomal membranes and increasing melanin content in the cells. Overall, we report the impact of cellular factors in prolonging drug retention and release from melanin-containing cells. These data and simulations will facilitate the design of melanin binding drugs with prolonged ocular actions.

Retraction: Hellinen et al. Retinal Pigment Epithelial Cell Line with Fast Differentiation and Improved Barrier Properties. Pharmaceutics 2019, 11, 412.

The published article [...].

Ocular metabolism and distribution of drugs in the rabbit eye: Quantitative assessment after intracameral and intravitreal administrations.

Quantitation of ocular drug metabolism is important, but only sparse data is currently available. Herein, the pharmacokinetics of four drugs, substrates of metabolizing enzymes, was investigated in albino rabbit eyes after intracameral and intravitreal administrations. Acetaminophen, brimonidine, cefuroxime axetil, and sunitinib and their corresponding metabolites were quantitated in the cornea, iris-ciliary body, aqueous humor, lens, vitreous humor, and neural retina with LC-MS/MS analytics. Non-compartmental analysis was employed to estimate the pharmacokinetic parameters of the parent drugs and metabolites. The area under the curve (AUC) values of metabolites were 12-70 times lower than the AUC values of the parent drugs in the tissues with the highest enzymatic activity. The ester prodrug cefuroxime axetil was an exception because it was efficiently and quantitatively converted to cefuroxime in the ocular tissues. In contrast to the liver, sulfotransferases, aldehyde oxidase, and cytochrome P450 3A activities were low in the eye and they had negligible impact on ocular drug clearance. With the exception of esterase substrates, metabolism seems to be a minor player in ocular pharmacokinetics. However, metabolites might contribute to ocular toxicity, and drug metabolism in various eye tissues should be investigated and understood thoroughly.

Ocular pharmacokinetics of atenolol, timolol and betaxolol cocktail: Tissue exposures in the rabbit eye.

Quantitative understanding of pharmacokinetics of topically applied ocular drugs requires more research to further understanding and to eventually allow predictive in silico models to be developed. To this end, a topical cocktail of betaxolol, timolol and atenolol was instilled on albino rabbit eyes. Tear fluid, corneal epithelium, corneal stroma with endothelium, bulbar conjunctiva, anterior sclera, iris-ciliary body, lens and vitreous samples were collected and analysed using LC-MS/MS. Iris-ciliary body was also analysed after intracameral cocktail injection. Non-compartmental analysis was utilized to estimate the pharmacokinetics parameters. The most lipophilic drug, betaxolol, presented the highest exposure in all tissues except for tear fluid after topical administration, followed by timolol and atenolol. For all drugs, iris-ciliary body concentrations were higher than that of the aqueous humor. After topical instillation the most hydrophilic drug, atenolol, had 3.7 times higher AUCiris-ciliary body than AUCaqueous humor, whereas the difference was 1.4 and 1.6 times for timolol and betaxolol, respectively. This suggests that the non-corneal route (conjunctival-scleral) was dominating the absorption of atenolol, while the corneal route was more important for timolol and betaxolol. The presented data increase understanding of ocular pharmacokinetics of a cocktail of drugs and provide data that can be used for quantitative modeling and simulation.

Biopharmaceutics of Topical Ophthalmic Suspensions: Importance of Viscosity and Particle Size in Ocular Absorption of Indomethacin.

Eye drops of poorly soluble drugs are frequently formulated as suspensions. Bioavailability of suspended drug depends on the retention and dissolution of drug particles in the tear fluid, but these factors are still poorly understood. We investigated seven ocular indomethacin suspensions (experimental suspensions with two particle sizes and three viscosities, one commercial suspension) in physical and biological tests. The median particle size (d50) categories of the experimental suspensions were 0.37-1.33 and 3.12-3.50 µm and their viscosity levels were 1.3, 7.0, and 15 mPa·s. Smaller particle size facilitated ocular absorption of indomethacin to the aqueous humor of albino rabbits. In aqueous humor the AUC values of indomethacin suspensions with different particle sizes, but equal viscosity, differed over a 1.5 to 2.3-fold range. Higher viscosity increased ocular absorption 3.4-4.3-fold for the suspensions with similar particle sizes. Overall, the bioavailability range for the suspensions was about 8-fold. Instillation of larger particles resulted in higher tear fluid AUC values of total indomethacin (suspended and dissolved) as compared to application of smaller particles. Despite these tear fluid AUC values of total indomethacin, instillation of the larger particles resulted in smaller AUC levels of indomethacin in the aqueous humor. This suggests that the small particles yielded higher concentrations of dissolved indomethacin in the tear fluid, thereby leading to improved ocular bioavailability. This new conclusion was supported by ocular pharmacokinetic modeling. Both particle size and viscosity have a significant impact on drug concentrations in the tear fluid and ocular drug bioavailability from topical suspensions. Viscosity and particle size are the key players in the complex interplay of drug retention and dissolution in the tear fluid, thereby defining ocular drug absorption and bioequivalence of ocular suspensions.

Microscale Thermophoresis as a Screening Tool to Predict Melanin Binding of Drugs.

Interactions between drugs and melanin pigment may have major impacts on pharmacokinetics. Therefore, melanin binding can modify the efficacy and toxicity of medications in ophthalmic and other disease of pigmented tissues, such as melanoma. As melanin is present in many pigmented tissues in the human body, investigation of pigment binding is relevant in drug discovery and development. Conventionally, melanin binding assays have been performed using an equilibrium binding study followed by chemical analytics, such as LC/MS. This approach is laborious, relatively slow, and limited to facilities with high performance quantitation instrumentation. We present here a screening of melanin binding with label-free microscale thermophoresis (MST) that utilizes the natural autofluorescence of melanin. We determined equilibrium dissociation constants (Kd) of 11 model compounds with melanin nanoparticles. MST categorized the compounds into extreme (chloroquine, penicillin G), high (papaverine, levofloxacin, terazosin), intermediate (timolol, nadolol, quinidine, propranolol), and low melanin binders (atropine, methotrexate, diclofenac) and displayed good correlation with binding parameter values obtained with the conventional binding study and LC/MS analytics. Further, correlation was seen between predicted melanin binding in human retinal pigment epithelium and choroid (RPE-choroid) and Kd values obtained with MST. This method represents a useful and fast approach for classification of compounds regarding melanin binding. Thus, the method can be utilized in various fields, including drug discovery, pharmacokinetics, and toxicology.

Ocular Intracameral Pharmacokinetics for a Cocktail of Timolol, Betaxolol, and Atenolol in Rabbits.

The mechanisms of drug clearance from the aqueous humor are poorly defined. In this study, a cocktail approach was used to simultaneously determine the pharmacokinetics of three ß-blocker agents after intracameral (ic) injection into the rabbit eyes. Aqueous humor samples were collected and analyzed using LC-MS/MS to determine drug concentrations. Pharmacokinetic parameters were obtained using a compartmental fitting approach, and the estimated clearance, volume of distribution, and half-life values were the following: atenolol (6.44 µL/min, 687 µL, and 73.87 min), timolol (19.30 µL/min, 937 µL, and 33.64 min), and betaxolol (32.20 µL/min, 1421 µL, and 30.58 min). Increased compound lipophilicity (atenolol < timolol < betaxolol) resulted in higher clearance and volume of distributions in the aqueous humor. Clearance of timolol and betaxolol is about 10 times higher than the aqueous humor outflow, demonstrating the importance of other elimination routes (e.g., uptake to iris and ciliary body and subsequent elimination via blood flow).

Quantitative Protein Expression in the Human Retinal Pigment Epithelium: Comparison Between Apical and Basolateral Plasma Membranes With Emphasis on Transporters.

Purpose: Retinal pigment epithelium (RPE) limits the xenobiotic entry from the systemic blood stream to the eye. RPE surface transporters can be important in ocular drug distribution, but it has been unclear whether they are expressed on the apical, basal, or both cellular surfaces. In this paper, we provide quantitative comparison of apical and basolateral RPE surface proteomes. Methods: We separated the apical and basolateral membranes of differentiated human fetal RPE (hfRPE) cells by combining apical membrane peeling and sucrose density gradient centrifugation. The membrane fractions were analyzed with quantitative targeted absolute proteomics (QTAP) and sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) to reveal the membrane protein localization on the RPE cell surfaces. We quantitated 15 transporters in unfractionated RPE cells and scaled their expression to tissue level. Results: Several proteins involved in visual cycle, cell adhesion, and ion and nutrient transport were expressed on the hfRPE plasma membranes. Most drug transporters showed similar abundance on both RPE surfaces, whereas large neutral amino acids transporter 1 (LAT1), p-glycoprotein (P-gp), and monocarboxylate transporter 1 (MCT1) showed modest apical enrichment. Many solute carriers (SLC) that are potential prodrug targets were present on both cellular surfaces, whereas putative sodium-coupled neutral amino acid transporter 7 (SNAT7) and riboflavin transporter (RFT3) were enriched on the basolateral and sodium- and chloride-dependent neutral and basic amino acid transporter (ATB0+) on the apical membrane. Conclusions: Comprehensive quantitative information of the RPE surface proteomes was reported for the first time. The scientific community can use the data to further increase understanding of the RPE functions. In addition, we provide insights for transporter protein localization in the human RPE and the significance for ocular pharmacokinetics.

The Basis for Strain-Dependent Rat Aldehyde Dehydrogenase 1A7 (ALDH1A7) Gene Expression.

Aldehyde hydrogenases (ALDHs) belong to a large gene family involved in oxidation of both endogenous and exogenous compounds in mammalian tissues. Among ALDHs, the rat ALDH1A7 gene displays a curious strain dependence in phenobarbital (PB)-induced hepatic expression: the responsive RR strains exhibit induction of both ALDH1A7 and CYP2B mRNAs and activities, whereas the nonresponsive rr strains show induction of CYP2B only. Here, we investigated the responsiveness of ALDH1A1, ALDH1A7, CYP2B1, and CYP3A23 genes to prototypical P450 inducers, expression of nuclear receptors CAR and pregnane X receptor, and structure of the ALDH1A7 promoter in both rat strains. ALDH1A7 mRNA, associated protein and activity were strongly induced by PB and modestly induced by pregnenolone 16α-carbonitrile in the RR strain but negligibly in the rr strain, whereas induction of ALDH1A1 and P450 mRNAs was similar between the strains. Reporter gene and chromatin immunoprecipitation assays indicated that the loss of ALDH1A7 inducibility in the rr strain is profoundly linked with a 16-base pair deletion in the proximal promoter and inability of the upstream DNA sequences to recruit constitutive androstane receptor-retinoid X receptor heterodimers. SIGNIFICANCE STATEMENT: Genetic variation in rat ALDH1A7 promoter sequences underlie the large strain-dependent differences in expression and inducibility by phenobarbital of the aldehyde dehydrogenase activity. This finding has implications for the design and interpretation of pharmacological and toxicological studies on the effects and disposition of aldehydes.

Characterization of artificially re-pigmented ARPE-19 retinal pigment epithelial cell model.

Melanin pigment has a significant role in ocular pharmacokinetics, because many drugs bind at high extent to melanin in the retinal pigment epithelial cells. Most retinal pigment epithelial cell lines lack pigmentation and, therefore, we re-pigmented human ARPE-19 cells to generate a pigmented cell model. Melanosomes from porcine retinal pigment epithelium were isolated and co-incubated with ARPE-19 cells that spontaneously phagocytosed the melanosomes. Internalized melanosomes were functionally integrated to the cellular system as evidenced by correct translocation of cellular Rab27a protein to the melanosomal membranes. The pigmentation was retained during cell cultivation and the level of pigmentation can be controlled by altering the amount of administered melanosomes. We used these cells to study melanosomal uptake of six drugs. The uptake was negligible with low melanin-binders (methotrexate, diclofenac) whereas most of the high melanin-binders (propranolol, chloroquine) were extensively taken up by the melanosomes. This cell line can be used to model pigmentation of the retinal pigment epithelium, while maintaining the beneficial cell line characteristics, such as fast generation of cultures, low cost, long-term maintenance and good reproducibility. The model enables studies at normal and decreased levels of pigmentation to model different retinal conditions.

Retinal Pigment Epithelial Cell Line with Fast Differentiation and Improved Barrier Properties.

Retinal pigment epithelium (RPE) acts as an outer blood-retinal barrier that limits the access of circulating xenobiotics to the eye. In addition, the RPE limits posterior elimination of intravitreally injected drugs to circulation. Thus, permeation in the RPE has a significant effect on ocular pharmacokinetics. The RPE is also a potentially important drug target in age-related macular degeneration. Therefore, the cell models of the RPE are important tools in ocular drug development, but poor availability and problems in reproducibility limit the use of primary RPE cell cultures. Furthermore, the best and widely used human cell line ARPE19 requires specialized culture conditions and a long time for cellular differentiation. In this paper, we describe a cell population arisen from the ARPE19 culture, with fast differentiation and improved barrier properties. This cell line, LEPI, forms clear microvilli and rapidly displays RPE-like cobblestone morphology after subculture in simple culture conditions. The LEPI cells show RPE-specific functions and expression of RPE65, ezrin, and BEST1 proteins. On filter, the LEPI cells develop tighter barrier than the ex vivo bovine RPE-choroid: permeability coefficients of beta-blockers (atenolol, nadolol, timolol, pindolol, metoprolol, betaxolol) ranged from 0.4 × 10-6 cm/sec to 2.3 × 10-6 cm/sec depending on the drug lipophilicity. This rapidly differentiating cell line will be an asset in ocular studies since it is easily maintained, it grows and differentiates quickly and does not require specialized culture conditions for differentiation. Thus, this cell line is suitable for both small scale assays and high throughput screening in drug discovery and development.

Nucleic acid delivery to differentiated retinal pigment epithelial cells using cell-penetrating peptide as a carrier.

Nucleic acid delivery to the eye is a promising treatment strategy for many retinal disorders. In this manuscript, retinal gene delivery with non-coated and chondroitin sulphate (CS) coated amphipathic and cationic peptides was tested. The transfection and gene knockdown efficiencies were evaluated in different retinal pigment epithelial (RPE) cell models including both dividing and differentiated cells. In addition, the mobility of peptide-based gene delivery systems was examined in porcine vitreous by particle tracking analysis. The results indicate that amphipathic and cationic peptides are safe in vitro and are capable of high transgene expression and gene knockdown in dividing cells. We further demonstrate that incorporation of CS improves the efficiency of gene delivery of peptide-based systems. Most importantly, the transgene expression mediated by both non-coated and CS coated peptides was high in differentiated as well as in human primary RPE cells which are typically difficult to transfect. Coating of peptide-based gene delivery systems with CS improved diffusion in the vitreous and enhanced the stability of the polyplexes. The results indicate that a peptide-based system can be fine-tuned as a promising approach for retinal gene delivery.

Loss of NRF-2 and PGC-1α genes leads to retinal pigment epithelium damage resembling dry age-related macular degeneration.

Age-related macular degeneration (AMD) is a multi-factorial disease that is the leading cause of irreversible and severe vision loss in the developed countries. It has been suggested that the pathogenesis of dry AMD involves impaired protein degradation in retinal pigment epithelial cells (RPE). RPE cells are constantly exposed to oxidative stress that may lead to the accumulation of damaged cellular proteins, DNA and lipids and evoke tissue deterioration during the aging process. The ubiquitin-proteasome pathway and the lysosomal/autophagosomal pathway are the two major proteolytic systems in eukaryotic cells. NRF-2 (nuclear factor-erythroid 2-related factor-2) and PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1 alpha) are master transcription factors in the regulation of cellular detoxification. We investigated the role of NRF-2 and PGC-1α in the regulation of RPE cell structure and function by using global double knockout (dKO) mice. The NRF-2/PGC-1α dKO mice exhibited significant age-dependent RPE degeneration, accumulation of the oxidative stress marker, 4-HNE (4-hydroxynonenal), the endoplasmic reticulum stress markers GRP78 (glucose-regulated protein 78) and ATF4 (activating transcription factor 4), and damaged mitochondria. Moreover, levels of protein ubiquitination and autophagy markers p62/SQSTM1 (sequestosome 1), Beclin-1 and LC3B (microtubule associated protein 1 light chain 3 beta) were significantly increased together with the Iba-1 (ionized calcium binding adaptor molecule 1) mononuclear phagocyte marker and an enlargement of RPE size. These histopathological changes of RPE were accompanied by photoreceptor dysmorphology and vision loss as revealed by electroretinography. Consequently, these novel findings suggest that the NRF-2/PGC-1α dKO mouse is a valuable model for investigating the role of proteasomal and autophagy clearance in the RPE and in the development of dry AMD.

Implications of melanin binding in ocular drug delivery.

Pigmented ocular tissues contain melanin within the intracellular melanosomes. Drugs bind to melanin at varying extent that ranges from no binding to extensive binding. Binding may lead to drug accumulation to the pigmented tissues and prolonged drug retention in the melanin containing cells. Therefore, melanin binding is an important feature that affects ocular drug delivery and biodistribution, but this topic has not been reviewed since 1998. In this review, we present current knowledge on ocular melanin, melanosomes and binding of drugs to pigmented cells and tissues. In vitro, in vivo and in silico methods in the field were critically evaluated, because the literature in this field can be confusing if the reader does not properly understand the methodological aspects. Literature analysis includes a comprehensive table of literature data on melanin binding of drugs. Furthermore, we aimed to give some insights beyond the current literature by making a chemical structure based classification model for melanin binding of drugs and kinetic simulations that revealed significant interplay between melanin binding and drug permeability across the melanosomal and plasma membranes. Overall, more mechanistic and systematic research is needed before the impact of melanin binding on ocular drug delivery can be properly understood and predicted.

Melanin binding study of clinical drugs with cassette dosing and rapid equilibrium dialysis inserts.

Melanin pigment is a negatively charged polymer found in pigmented human tissues. In the eye, iris, ciliary body, choroid and retinal pigment epithelium (RPE) are heavily pigmented. Several drug molecules are known to bind to melanin, but larger sets of drugs have not been compared often in similar test conditions. In this study, we introduce a powerful tool for screening of melanin binding. The binding of a set of 34 compounds to isolated porcine RPE melanin was determined by cassette (n-in-one) dosing in rapid equilibrium dialysis inserts and the binding was quantitated with LC-MS/MS analytics. The compounds represented large variety in melanin binding (from 8.6%, ganciclovir) to over 95% bound (ampicillin and ciprofloxacin). The data provides information on melanin binding of small molecular weight compounds that are used for ocular (e.g. brinzolamide, ganciclovir) and systemic (e.g. tizanidine, indomethacin) therapy. Interestingly, competition among compounds was seen for melanin binding and the binding did not show any correlation with plasma protein binding. These results increase the understanding of melanin binding of ocular drugs and can be further exploited to predict pharmacokinetics in the eye. Pigment binding provides an interesting option for improved drug distribution to retina and choroid that are difficult target tissues in drug delivery.