Transporter Protein Expression of Corneal Epithelium in Rabbit and Porcine: Evaluation of Models for Ocular Drug Transport Study.

The transcorneal route is the main entry route for drugs to the intraocular parts, after topical administration. The outer surface, the corneal epithelium (CE), forms the rate-limiting barrier for drug permeability. Information about the role and protein expression of drug and amino acid transporter proteins in the CE is sparse and lacking. The aim of our study was to characterize transporter protein expression in rabbit and porcine CE to better understand potential drug and nutrient absorption after topical administration. Proteins, mainly Abc and Slc transporters, were characterized with quantitative targeted absolute proteomics and global untargeted proteomics methods. In the rabbit CE, 24 of 48 proteins were detected in the targeted approach, and 21 of these were quantified. In the porcine CE, 26 of 58 proteins were detected in the targeted approach, and 20 of these were quantified. Among these, 15 proteins were quantified in both animals: 4f2hc (Slc3a2), Aqp0, Asct1 (Slc1a4), Asct2 (Slc1a5), Glut1 (Slc2a1), Hmit (Slc2a13), Insr, Lat1 (Slc7a5), Mct1 (Slc16a1), Mct2 (Slc16a7), Mct4 (Slc16a3), Mrp 4 (Abcc4), Na+/K+-ATPase, Oatp3a1 (Slco3a1), and Snat2 (Slc38a2). Overall, the global proteomics results supported the targeted proteomics results. Organic anion transporting polypeptide Oatp3a1 was detected and quantified for the first time in both rabbit (1.4 ± 0.4 fmol/cm2) and porcine (11.1 ± 5.3 fmol/cm2) CE. High expression levels were observed for L-type amino acid transporter, Lat1, which was quantified with newly selected extracellular domain peptides in rabbit (48.9 ± 11.8 fmol/cm2) and porcine (37.6 ± 11.5 fmol/cm2) CE. The knowledge of transporter protein expression in ocular barriers is a key factor in the successful design of new ocular drugs, pharmacokinetic modeling, understanding ocular diseases, and the translation to human.

Barcode lipids for absolute quantitation of liposomes in ocular tissues.

Lipid-based drug formulations are promising systems for improving delivery of drugs to ocular tissues, such as retina. To develop lipid-based systems further, an improved understanding of their pharmacokinetics is required, but high-quality in vivo experiments require a large number of animals, raising ethical and economic questions. In order to expedite in vivo kinetic testing of lipid-based systems, we propose a barcode approach that is based on barcoding liposomes with non-endogenous lipids. We developed and evaluated a liquid-chromatography-mass spectrometry method to quantify many liposomes simultaneously in aqueous humor, vitreous, and neural retina at higher than ±20% precision and accuracy. Furthermore, we showed in vivo suitability of the method in pharmacokinetic evaluation of six different liposomes after their simultaneous injection into the rat vitreal cavity. We calculated pharmacokinetic parameters in vitreous and aqueous humor, quantified liposome concentrations in the retina, and quantitated retinal distribution of the liposomes in the rats. Compared to individual injections of the liposome formulations, the barcode-based study design enabled reduction of animal numbers from 72 to 12. We believe that the proposed approach is reliable and will reduce and refine ocular pharmacokinetic experiments with liposomes and other lipid-based systems.

Comprehensive ocular and systemic pharmacokinetics of dexamethasone after subconjunctival and intravenous injections in rabbits.

Even though subconjunctival injections are used in clinics, their quantitative pharmacokinetics has not been studied systematically. For this purpose, we evaluated the ocular and plasma pharmacokinetics of subconjunctival dexamethasone in rabbits. Intravenous injection was also given to enable a better understanding of the systemic pharmacokinetics. Dexamethasone concentrations in plasma (after subconjunctival and intravenous injections) and four ocular tissues (iris-ciliary body, aqueous humour, neural retina and vitreous) were analysed using LC-MS/MS. Population pharmacokinetic modelling for plasma data from both injection routes were used, and for first time the constant rate of absorption of dexamethasone from the subconjunctival space into plasma was estimated (ka,plasma = 0.043 min-1, i.e. absorption half-life of 17.3 min). Non-compartmental analysis was used for the ocular data analysis and resulting in ocular drug exposure of iris-ciliary body (AUC0-∞= 41984 min·ng/g) > neural retina (AUC0-∞= 25511 min·ng/g) > vitreous (AUC0-∞= 7319 min·ng/mL) > aqueous humour (AUC0-∞= 6146 min·ng/mL). The absolute bioavailability values after subconjunctival injection, reported for the first time, were 0.74 % in aqueous humour (comparable to topical dexamethasone suspensions), and 0.30 % in vitreous humour (estimated to be higher than in topical administration). These novel and comprehensive pharmacokinetic data provide valuable information on the potential for exploiting this route in ocular drug development for treating both, anterior and posterior segment ocular diseases. Moreover, the new generated dexamethasone-parameters are a step-forward in building predictive pharmacokinetic models to support the design of new subconjunctival dexamethasone formulations, which may sustain drug effect for longer period of time.

The prophylactic value of TNF-α inhibitors against retinal cell apoptosis and optic nerve axon loss after corneal surgery or trauma.

BACKGROUND AND PURPOSE: Late secondary glaucoma is an often-severe complication after acute events like anterior segment surgery, trauma and infection. TNF-α is a major mediator that is rapidly upregulated, diffusing also to the retina and causes apoptosis of the ganglion cells and degeneration of their optic nerve axons (mediating steps to glaucomatous damage). Anti-TNF-α antibodies are in animals very effective in protecting the retinal cells and the optic nerve-and might therefore be useful prophylactically against secondary glaucoma in future such patients. Here we evaluate (1) toxicity and (2) efficacy of two TNF-α inhibitors (adalimumab and infliximab), in rabbits by subconjunctival administration. METHODS: For drug toxicity, animals with normal, unburned corneas were injected with adalimumab (0.4, 4, or 40 mg), or infliximab (1, 10, or 100 mg). For drug efficacy, other animals were subjected to alkali burn before such injection, or steroids (for control). The rabbits were evaluated clinically with slit lamp and photography, electroretinography, optical coherence tomography, and intraocular pressure manometry. A sub-set of eyes were stained ex vivo after 3 days for retinal cell apoptosis (TUNEL). In other experiments the optic nerves were evaluated by paraphenylenediamine staining after 50 or 90 days. Loss of retinal cells and optic nerve degeneration were quantified. RESULTS: Subconjunctival administration of 0.4 mg or 4.0 mg adalimumab were well tolerated, whereas 40.0 mg was toxic to the retina. 1, 10, or 100 mg infliximab were also well tolerated. Analysis of the optic nerve axons after 50 days confirmed the safety of 4.0 mg adalimumab and of 100 mg infliximab. For efficacy, 4.0 mg adalimumab subconjunctivally in 0.08 mL provided practically full protection against retinal cell apoptosis 3 days following alkali burn, and infliximab 100 mg only slightly less. At 90 days following burn injury, control optic nerves showed about 50% axon loss as compared to 8% in the adalimumab treatment group. CONCLUSIONS: Subconjunctival injection of 4.0 mg adalimumab in rabbits shows no eye toxicity and provides excellent neuroprotection, both short (3 days) and long-term (90 days). Our total. accumulated data from several of our studies, combined with the present paper, suggest that corneal injuries, including surgery, might benefit from routine administration of anti-TNF-α biologics to reduce inflammation and future secondary glaucoma.

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.

Quantitative intravitreal pharmacokinetics in mouse as a step towards inter-species translation.

Although mouse models are widely used in retinal drug development, pharmacokinetics in mouse eye is poorly understood. In this study, we applied non-invasive in vivo fluorophotometry to study pharmacokinetics of intravitreal fluorescein sodium (molecular weight 0.38 kDa) and fluorescein isothiocyanate-dextran (FD-150; molecular weight 150 kDa) in mice. Intravitreal half-lives of fluorescein and FD-150 in mouse eyes were 0.53 ± 0.06 h and 2.61 ± 0.86 h, respectively. These values are 8-230 times shorter than the elimination half-lives of similar compounds in the human vitreous. The apparent volumes of distribution in the mouse vitreous were close to the anatomical volume of the mouse vitreous (FD-150, 5.1 µl; fluorescein, 9.6 µl). Dose scaling factors were calculated from mouse to rat, rabbit, monkey and human translation. Based on pharmacokinetic modelling and compound concentrations in the vitreous and anterior chamber, fluorescein is mainly eliminated posteriorly across blood-retina barrier, but FD-150 is cleared via aqueous humour outflow. The results of this study improve the knowledge of intravitreal pharmacokinetics in mouse and facilitate inter-species scaling in ocular drug development.

Mass spectrometry in ocular drug research.

Mass spectrometry (MS) has been proven as an excellent tool in ocular drug research allowing analyzes from small samples and low concentrations. This review begins with a short introduction to eye physiology and ocular pharmacokinetics and the relevance of advancing ophthalmic treatments. The second part of the review consists of an introduction to ocular proteomics, with special emphasis on targeted absolute quantitation of membrane transporters and metabolizing enzymes. The third part of the review deals with liquid chromatography-MS (LC-MS) and MS imaging (MSI) methods used in the analysis of drugs and metabolites in ocular samples. The sensitivity and speed of LC-MS make simultaneous quantitation of various drugs and metabolites possible in minute tissue samples, even though ocular sample preparation requires careful handling. The MSI methodology is on the verge of becoming as important as LC-MS in ocular pharmacokinetic studies, since the spatial resolution has reached the level, where cell layers can be separated, and quantitation with isotope-labeled standards has come more reliable. MS will remain in the foreseeable future as the main analytical method that will progress our understanding of ocular pharmacokinetics.

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.

Topical pharmacokinetics of brinzolamide suspensions in rabbits and variability analysis for sample size and design considerations.

Identifying critical attributes for complex locally acting ophthalmic formulations and establishing in vitro-in vivo correlations can facilitate selection of appropriate thresholds for formulation changes that reflect lack of impact on in vivo performance. In this study the marketed antiglaucoma product Azopt® (1% brinzolamide suspension) and five other brinzolamide formulations varying in particle size distributions and apparent viscosities were topically administered in rabbits, and their ocular pharmacokinetics was determined in multiple ocular tissues. Statistical evaluation with ANOVA showed no significant differences between the formulations in the peak drug concentration (Cmax) in the aqueous humor and iris-ciliary body. As a post-hoc analysis, the within animal and total variability was determined for Cmax in the aqueous humor and iris-ciliary body. Based on the observed variability, we investigated the sample size needed for two types of study designs to observe statistically significant differences in Cmax. For the sample size calculations, assuming both 25% and 50% true differences in Cmax between two formulations, two study designs were compared: paired-eye dosing design (one formulation in one eye and another formulation in the other eye of the same animal at the same time) versus parallel-group design. The number of rabbits needed in the paired-eye dosing design are much lower than in the parallel-group design. For example, when the true difference in aqueous humor Cmax is 25%, nine rabbits are required in the paired-eye design versus seventy rabbits (35 per treatment) in the parallel-group design to observe a statistically significant difference with a power of 80%. Therefore, the proposed paired-eye dosing design is a viable option for the design of pharmacokinetic studies comparing ophthalmic products to determine the impact of formulation differences.

Tailoring surface properties of liposomes for dexamethasone intraocular administration.

Diseases of the posterior eye segment are often characterized by intraocular inflammation, which causes, in the long term, severe impairment of eye functions and, ultimately, vision loss. Aimed at enhancing the delivery of anti-inflammatory drugs to the posterior eye segment upon intravitreal administration, we developed liposomes with an engineered surface to control their diffusivity in the vitreous and retina association. Hydrogenated soybean phosphatidylcholine (HSPC)/cholesterol liposomes were coated with (agmatinyl)6-maltotriosyl-acetamido-N-(octadec-9-en-1-yl)hexanamide (Agm6-M-Oleate), a synthetic non-peptidic cell penetration enhancer (CPE), and/or 5% of mPEG2kDa-DSPE. The zeta potential of liposomes increased, and the mobility in bovine vitreous and colloidal stability decreased with the Agm6-M-Oleate coating concentration. Oppositely, mPEG2kDa-DSPE decreased the zeta potential of liposomes and restored both the diffusivity and the stability in vitreous. Liposomes with 5 mol% Agm6-M-Oleate coating were well tolerated by ARPE-19 retina cells either with or without mPEG2kDa-DSPE, while 10 mol% Agm6-M-Oleate showed cytotoxicity. Agm6-M-Oleate promoted the association of liposomes to ARPE-19 cells with respect to plain liposomes, while mPEG2kDa-DSPE slightly reduced the cell interaction. Dexamethasone hemisuccinate (DH) was remotely loaded into liposomes with a loading capacity of ∼10 wt/wt%. Interestingly, mPEG2kDa-DSPE coating reduced the rate of DH release and enhanced the disposition of Agm6-M-Oleate coated liposomes in the ARPE-19 cell cytosol resulting in a more efficient anti-inflammatory effect. Finally, mPEG2kDa-DSPE enhanced the association of DH-loaded Agm6-M-Oleate coated liposomes to explanted rat retina, which reflected in higher viability of inner and outer nuclear layer cells.

Activity and Expression of Carboxylesterases and Arylacetamide Deacetylase in Human Ocular Tissues.

As a multitissue organ, the eye possesses unique anatomy and physiology, including differential expression of drug-metabolizing enzymes. Several hydrolytic enzymes that play a major role in drug metabolism and bioactivation of prodrugs have been detected in ocular tissues, but data on their quantitative expression is scarce. Also, many ophthalmic drugs are prone to hydrolysis. Metabolic characterization of individual ocular tissues is useful for the drug development process, and therefore, seven individual ocular tissues from human eyes were analyzed for the activity and expression of carboxylesterases (CESs) and arylacetamide deacetylase (AADAC). Generic and selective human esterase substrates 4-nitrophenyl acetate (most esterases), D-luciferin methyl ester (CES1), fluorescein diacetate and procaine (CES2), and phenacetin (AADAC) were applied to determine the enzymes' specific activities. Enzyme kinetics and inhibition studies were performed with isoform-selective inhibitors digitonin (CES1) and verapamil and diltiazem (CES2). Enzyme contents were determined using quantitative targeted proteomics, and CES2 expression was confirmed by western blotting. The expression and activity of human CES1 among ocular tissues varied by >10-fold, with the highest levels found in the retina and iris-ciliary body. In contrast, human CES2 expression appeared lower and more similar between tissues, whereas AADAC could not be detected. Inhibition studies showed that hydrolysis of fluorescein diacetate is also catalyzed by enzymes other than CES2. This study provides, for the first time, quantitative information on the tissue-dependent expression of human ocular esterases, which can be useful for the development of ocular drugs, prodrugs, and in pharmacokinetic modeling of the eye. SIGNIFICANCE STATEMENT: Novel and comprehensive data on the protein expression and activities of carboxylesterases from individual human eye tissues are generated. In combination with previous reports on preclinical species, this study will improve the understanding of interspecies differences in ocular drug metabolism and aid the development of ocular pharmacokinetics models.

Antiangiogenic AAV2 gene therapy with a truncated form of soluble VEGFR-2 reduces the growth of choroidal neovascularization in mice after intravitreal injection.

Pathological angiogenesis related to neovascularization in the eye is mediated through vascular endothelial growth factors (VEGFs) and their receptors. Ocular neovascular-related diseases are mainly treated with anti-VEGF agents. In this study we evaluated the efficacy and safety of novel gene therapy using adeno associated virus 2 vector expressing a truncated form of soluble VEGF receptor-2 fused to the Fc-part of human IgG1 (AAV2-sVEGFR-2-Fc) to inhibit ocular neovascularization in laser induced choroidal neovascularization (CNV) in mice. The biological activity of sVEGFR-2-Fc was determined in vitro. It was shown that sVEGFR-2-Fc secreted from ARPE-19 cells was able to bind to VEGF-A165 and reduce VEGF-A165 induced cell growth and survival. A single intravitreal injection (IVT) of AAV2-sVEGFR-2-Fc (1 µl, 4.7 × 1012 vg/ml) one-month prior laser photocoagulation did not cause any changes in the retinal morphology and significantly suppressed fluorescein leakage at 7, 14, 21 and 28 days post-lasering compared to controls. Macrophage infiltration was observed after the injection of both AAV2-sVEGFR-2-Fc and PBS. Our findings indicate that AAV2 mediated gene delivery of the sVEGFR-2-Fc efficiently reduces formation of CNV and could be developed to a therapeutic tool for the treatment of retinal diseases associated with neovascularization.

Swarms of chemically modified antiviral siRNA targeting herpes simplex virus infection in human corneal epithelial cells.

Herpes simplex virus type 1 (HSV-1) is a common virus of mankind and HSV-1 infections are a significant cause of blindness. The current antiviral treatment of herpes infection relies on acyclovir and related compounds. However, acyclovir resistance emerges especially in the long term prophylactic treatment that is required for prevention of recurrent herpes keratitis. Earlier we have established antiviral siRNA swarms, targeting sequences of essential genes of HSV, as effective means of silencing the replication of HSV in vitro or in vivo. In this study, we show the antiviral efficacy of 2´-fluoro modified antiviral siRNA swarms against HSV-1 in human corneal epithelial cells (HCE). We studied HCE for innate immunity responses to HSV-1, to immunostimulatory cytotoxic double stranded RNA, and to the antiviral siRNA swarms, with or without a viral challenge. The panel of studied innate responses included interferon beta, lambda 1, interferon stimulated gene 54, human myxovirus resistance protein A, human myxovirus resistance protein B, toll-like receptor 3 and interferon kappa. Our results demonstrated that HCE cells are a suitable model to study antiviral RNAi efficacy and safety in vitro. In HCE cells, the antiviral siRNA swarms targeting the HSV UL29 gene and harboring 2´-fluoro modifications, were well tolerated, induced only modest innate immunity responses, and were highly antiviral with more than 99% inhibition of viral release. The antiviral effect of the 2'-fluoro modified swarm was more apparent than that of the unmodified antiviral siRNA swarm. Our results encourage further research in vitro and in vivo on antiviral siRNA swarm therapy of corneal HSV infection, especially with modified siRNA swarms.

Improved ocular delivery of quercetin and resveratrol: A comparative study between binary and ternary cyclodextrin complexes.

The number of patients affected by Dry Eye Disease (DED) had notably increased worldwide, addressing the need of novel therapeutic approaches. Polyphenols, quercetin (QUE) and resveratrol (RSV) show necessary antioxidant and anti-inflammatory properties to manage DED, but their application as topical eyedrops is restricted by low aqueous solubility and low chemical stability. Cyclodextrins (CD) are widely used to improve physicochemical characteristics of drugs. Consequently, the aim of this study was to make a comparison between binary complexes with quercetin, resveratrol and cyclodextrins and tertiary complexes adding hyaluronic acid (HA). Both complexes were able to enhance solubility and stability of QUE and RSV. AFM imaging and DLS measurements disclose the formation of spherical nanoaggregates within tertiary complexes of both QUE and RSV with mean diameters of 103 and 82 nm. Neither complex demonstrated cytotoxic effect in in vitro studies in corneal (HCE) and conjunctival (IM-ConjEpi) cell lines. In HCE cells, complexes containing QUE or RSV at their highest concentrations were able to scavenge more than 95 % of the ROS that were produced intracellularly (p < 0.005). Similar response was observed with IM-ConjEpi cells. The antioxidant effect was maintained in the complexes with HA. This confirmed their potential as viable topical treatment for DED.

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.

Quantitative pharmacokinetic analyses of anterior and posterior elimination routes of intravitreal anti-VEGF macromolecules using published human and rabbit data.

The purpose of this study was to evaluate the contribution of the anterior elimination route for four anti-vascular endothelial growth factor (anti-VEGF) macromolecules (aflibercept, bevacizumab, pegaptanib and ranibizumab) after intravitreal injection using published human and rabbit data and three previously described pharmacokinetic (PK) modeling methods. A PubMed search was used to identify published studies with concentration-time data. The data were utilized only if the intravitreally injected drugs were used as plain solutions and several criteria for a well-performed PK study were fulfilled. The three methods to analyze rabbit data were (1) the equation for vitreal elimination half-life based molecular size assuming anterior elimination, (2) Maurice equation and plot for the ratio of aqueous humor (AH) to vitreal concentration assuming anterior elimination, and (3) the equation for amount of macromolecule eliminated anteriorly based on the area under the curve in AH. The first and third methods were used for human data. In the second and third methods, AH flow rate is a key model parameter, and it was varied between 2 and 3 µl/min. The methods were applied to data from 9 rabbit studies (1 for aflibercept, 5 for bevacizumab, and 3 for ranibizumab) and 5 human studies (1 for aflibercept, 3 for bevacizumab, and 1 for ranibizumab). Experimental half-lives of anti-VEGF macromolecules in both vitreous and aqueous humor were close to those calculated with the equations for vitreal elimination half-life in humans and rabbits. Rabbit data analyzed with Maurice plot indicated that the contribution of anterior elimination was usually at least 75%. In most human and rabbit studies, the calculated percentage of anterior elimination was at least 51%. Variability between studies was extensive for bevacizumab and ranibizumab. The results suggest that the anterior elimination route dominates after intravitreal injection of anti-VEGF macromolecules. However, the clinical data are sparse and variability is extensive, the latter emphasizing the need of proper experimental design.

Imaging, quantitation and kinetic modelling of intravitreal nanomaterials.

In this study, the intravitreal pharmacokinetics of nanomaterials were investigated in vivo in rats and rabbits. Impact of particle size and shape (spherical, longitudinal) on ocular particle distribution and elimination was investigated with fundus camera, optical coherence tomography and ocular fluorophotometry. Differently sized particles showed prolonged ocular retention and remarkable differences in vitreal elimination, but size dependence was consistent, suggesting that other features have influence on their vitreal kinetics. We also demonstrate that liposomes are eliminated from the rabbit vitreous mainly via the anterior route. Simulation of drug concentrations after injection of intravitreal particles shows the importance of synchronized particle retention and drug release rate for efficient drug delivery. In conclusion, we provide kinetic insights in intravitreally administered nanoparticles to improve retinal drug delivery.

Liposomal sunitinib for ocular drug delivery: A potential treatment for choroidal neovascularization.

Choroidal neovascularization (CNV) is a prevalent vision-threatening vascular disorder in aging population. CNV is associated with several diseases in the posterior segment of the eye such as age-related macular degeneration (AMD). In this study we developed sunitinib-loaded liposomes to block the neovascularization signalling pathway through inhibition of tyrosine kinase of vascular endothelial growth factor receptors (VEGFRs). Liposomal sunitinib formulations were prepared by thin film hydration method and studied for their encapsulation efficiency (EE), loading capacity (LC) and drug release profile in buffer andvitreous. Our finding showed that the liposomes (mean size 104 nm) could effectively entrap sunitinib (EE ≈ 95%) at relatively high loading capacity (LC ≈ 5%) and release sunitinib over at least 3 days. Intravitreal sunitinib-loaded liposomes revealed inhibitory effect on established neovascularization in laser-induced CNV mouse model while the intravitreal injection of sunitinib solubilized with cyclodextrin was inefficient in management of neovascularization. Accordingly, liposomal sunitinib is a promising drug delivery system that should be further studied to inhibit the CNV related to AMD.