Ocular Drug Development Research Articles

Physiologically based pharmacokinetic models for systemic disposition of protein therapeutics in rabbits.

Physiologically based pharmacokinetic (PBPK) modelling is an important tool to predict drug disposition in the body. Rabbits play a pivotal role as a highly valued small animal model, particularly in the field of ocular therapeutics, where they serve as a crucial link between preclinical research and clinical applications. In this context, we have developed PBPK models designed specifically for rabbits, with a focus on accurately predicting the pharmacokinetic profiles of protein therapeutics following intravenous administration. Our goal was to comprehend the influence of key physiological factors on systemic disposition of antibodies and their functional derivatives. For the development of the systemic PBPK models, rabbit physiological factors such as gene expression, body weight, neonatal fragment crystallizable receptor (FcRn) binding, target binding, target concentrations, and target turnover rate were meticulously considered. Additionally, key protein parameters, encompassing hydrodynamic radius, binding kinetic constants (KD, koff), internal degradation of the protein-target complex, and renal clearance, were represented in the models. Our final rabbit models demonstrated a robust correlation between predicted and observed serum concentration-time profiles after single intravenous administration in rabbits, covering IgG, Fab, F(ab)2, Fc, and Fc fusion proteins from various publications. These pharmacokinetic simulations offer a promising platform for translating preclinical findings to clinical settings. The presented rabbit intravenous PBPK models lay an important foundation for more specific applications of protein therapeutics in ocular drug development.

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.

Profiling tear film enzymes reveals major metabolic pathways involved in the homeostasis of the ocular surface

The ocular surface (OS) enzymes are of great interest due to their potential for novel ocular drug development. We aimed first to profile and classify the enzymes of the OS to describe major biological processes and pathways that are involved in the maintenance of homeostasis. Second, we aimed to compare the enzymatic profiles between the two most common tear collection methods, capillary tubes (CT) and Schirmer strips (ScS). A comprehensive tear proteomic dataset was generated by pooling all enzymes identified from nine tear proteomic analyses of healthy subjects using mass spectrometry. In these studies, tear fluid was collected using CT (n = 4), ScS (n = 4) or both collection methods (n = 1). Classification and functional analysis of the enzymes was performed using a combination of bioinformatic tools. The dataset generated identified 1010 enzymes. The most representative classes were hydrolases (EC 3) and transferases (EC 2). Phosphotransferases, esterases and peptidases were the most represented subclasses. A large portion of the identified enzymes was common to both collection methods (n = 499). More enzymes were specifically detected in the ScS-extracted proteome. The major pathways in which the identified enzymes participate are related to the immune system and protein, carbohydrate and lipid metabolism. Metabolic processes for nucleosides, cellular amides, sugars and sulfur compounds constituted the most enriched biological processes. Knowledge of these molecules highly susceptible to pharmacological manipulation might help to predict the metabolism of ophthalmic medications and develop novel prodrug strategies as well as new drug delivery systems. Combining such extensive knowledge of the OS enzymes with new analytical approaches and techniques might create new prospects for understanding, predicting and manipulating the metabolism of ocular pharmaceuticals. Our study reports new, essential data on OS enzymes while also comparing the enzyme profiles obtained via the two most popular methods of tear collection, capillary tubes and Schirmer strips.

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.

Implantable sustained-release drug delivery systems: a revolution for ocular therapeutics.

Due to the inimitable anatomical structure of the eyeball and various physiological barriers, conventional ocular local administration is often complicated by apparent shortcomings, such as limited bioavailability and short drug retention. Thus, developing methods for sustainable, safe and efficient drug delivery to ocular target sites has long been an urgent need. This study briefly summarizes the barriers to ocular drug administration and various ocular drug delivery routes and highlights recent progress in ocular implantable sustained-release drug delivery systems (DDSs) to provide literature evidence for developing novel ocular implants for sustained drug delivery. We conducted a comprehensive search of studies on ocular implantable sustained-release DDSs in PubMed and Web of Science using the following keywords: ocular, implantable and drug delivery system. More than 400 papers were extracted. Publications focused on sustained and controlled drug release were primarily considered. Experimental articles involving DDSs that cannot be implanted into the eye through surgeries and cannot be inserted into ocular tissues in solid form were excluded. Approximately 143 publications were reviewed to summarize the most current information on the subject. In recent years, numerous ocular sustained-release DDSs using lipids, nanoparticles and hydrogels as carriers have emerged. With unique properties and systematic design, ocular implantable sustained-release DDSs are able to continuously maintain drug release, effectively sustain the therapeutic concentration in target tissues, and substantially enhance the therapeutic efficacy. Nevertheless, few ocular implantable sustained-release DDSs have been available in clinical use. Ocular implantable sustained-release DDSs have become a new focus in the field of ocular drug development through unique designs and improvements in the materials of drug carriers, administration methods and dosage forms. With more ocular implantable sustained-release DDSs being commercialized, ocular therapeutics may be revolutionized.

Immunology and Pathology in Ocular Drug Development.

Clear vision is dependent on features that protect the anatomical integrity of the eye (cornea and sclera) and those that contribute to internal ocular homeostasis by conferring hemangiogenic (avascular tissues and antiangiogenic factors), lymphangiogenic (lack of draining lymphatics), and immunologic (tight junctions that form blood-ocular barriers, immunosuppressive cells, and modulators) privileges. The later examples are necessary components that enable the eye to maintain an immunosuppressive environment that responds to foreign invaders in a deviated manner, minimizing destructive inflammation that would impair vision. These conditions allowed for the observations made by Medawar, in 1948, of delayed rejection of allogenic tissue grafts in the anterior chamber of mouse eye and permit the sequestration of foreign invaders (eg, Toxoplasma gondii) within the retina of healthy individuals. Yet successful development of intraocular drugs (biologics and delivery devices) has been stymied by adverse ocular pathology, much of which is driven by immune pathways. The eye can be intolerant of foreign protein irrespective of delivery route, and endogenous ocular cells have remarkable plasticity when recruited to preserve visual function. This article provides a review of current understanding of ocular immunology and the potential role of immune mechanisms in pathology observed with intraocular drug delivery.

Introduction to Special Issue on Ocular Pathology and Drug Development.

Dysfunction of the visual system remains a leading cause of human disability worldwide. Preclinical studies are a key component of efforts to develop drugs and devices to ameliorate visual impairment. Although new opportunities for the delivery of targeted ocular therapeutics have been created, clinical success has been confounded by unique challenges of drug development for the eye. This Special Issue brings together a broad range of articles that augment our current understanding of the visual system and highlight methods for assessing ocular toxicity and some of the current challenges in ocular drug development. Topics addressed include the anatomy, developmental anatomy, and/or immunobiology of the visual system and associated lymphoid tissues; animal models; methods for assessing ocular toxicity; spontaneous background and procedure-related microscopic findings and common artifacts in histologic sections of ocular tissues; and novel ocular drug delivery systems.

Quantification of Drugs in Distinctly Separated Ocular Substructures of Albino and Pigmented Rats.

The rat is a commonly used species in ocular drug research. Detailed methods of separating rat ocular tissues have not been described in literature. To understand the intraocular drug distribution, we developed a robust method for the separation of individual anterior and posterior substructures of pigmented Brown Norway (BN) and albino Wistar Han (WH) rat eyes, followed by quantification of drug concentration in these substructures. A short formalin incubation, which did not interfere with drug quantification, enabled the preservation of individual tissue sections while minimizing cross-tissue contamination, as demonstrated by histological analysis. Following oral administration, we applied the tissue separation method, in order to determine the ocular concentrations of dexamethasone and levofloxacin, as well as two in-house molecules BI 113823 and BI 1026706, compounds differing in their melanin binding. The inter-individual variability in tissue partitioning coefficients (Kp) was low, demonstrating the reproducibility of the separation method. Kp values of individual tissues varied up to 100-fold in WH and up to 46,000-fold in BN rats highlighting the importance of measuring concentration directly from the ocular tissue of interest. Additionally, clear differences were observed in the BN rat tissue partitioning compared to the WH rat. Overall, the developed method enables a reliable determination of small molecule drug concentrations in ocular tissues to support ocular drug research and development.

Intravitreal Pharmacokinetics in Mice: SPECT/CT Imaging and Scaling to Rabbits and Humans.

Preclinical in vivo tests of retinal drug responses are carried out in mice and rats, often after intravitreal injections. However, quantitative pharmacokinetics in the mouse eye is poorly understood. Ocular pharmacokinetics studies are usually done in rabbits. We investigated elimination of three compounds ([99mTc]Tc-pentetate, [111In]In-pentetreotide, [99mTc]Tc-human serum albumin with molecular weights of 510.2 Da, 1506.4 Da, and 66.5 kDa, respectively) from mouse vitreous using imaging with single photon emission computed tomography/computed tomography (SPECT/CT). Increasing molecular weight decreased elimination of the compounds from the mouse eyes. Half-lives of [99mTc]Tc-pentetate, [111In]In-pentetreotide, and [99mTc]Tc-human serum albumin in the mouse eyes were 1.8 ± 0.5 h, 4.3 ± 1.7 h, and 30.0 ± 9.0 h, respectively. These values are 3-12-fold shorter than half-lives of similar compounds in the rabbit vitreous. Dose scaling factors were calculated for mouse-to-rabbit and mouse-to-man translation. They were 27-90 and 38-126, respectively, for intravitreal injections in rabbit and man. We show ocular pharmacokinetic parameters for mice and interspecies scaling factors that may augment ocular drug discovery and development.

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.

Imaging Techniques in the Diagnosis and Management of Ocular Tumors: Prospects and Challenges.

Different types of imaging modalities are used in the diagnosis of ocular cancer. Selection of an imaging modality is based on the features of a tumor as well as the inherent characteristics of the imaging technique. It is vital to select an appropriate imaging modality in diagnosis of ocular tumor with confidence. This review focuses on five most commonly used imaging modalities, i.e., positron emission tomography-computed tomography (PET/CT), single photon emission computed tomography (SPECT), optical coherence tomography (OCT), ultrasound (US), and magnetic resonance imaging (MRI). The principal of imaging modalities is briefly explained, along with their role in the diagnosis and management of the most common ocular tumors such as retinoblastoma and uveal melanoma. Further, the diagnostic features of ocular tumors corresponding to each imaging modality and possibilities of utilizing imaging techniques in the process of ocular drug development are included in this review.

The SPOTS System: An Ocular Scoring System Optimized for Use in Modern Preclinical Drug Development and Toxicology.

To present a semiquantitative ocular scoring system comprising elements and criteria that address many of the limitations associated with systems commonly used in preclinical studies, providing enhanced cross-species applicability and predictive value in modern ocular drug and device development. Revisions to the ocular scoring systems of McDonald-Shadduck and Hackett-McDonald were conducted by board-certified veterinary ophthalmologists at Ocular Services On Demand (OSOD) over the execution of hundreds of in vivo preclinical ocular drug and device development studies and general toxicological investigations. This semiquantitative preclinical ocular toxicology scoring (SPOTS) system was driven by limitations of previously published systems identified by our group's recent review of slit lamp-based scoring systems in clinical ophthalmology, toxicology, and vision science. The SPOTS system provides scoring criteria for the anterior segment, posterior segment, and characterization of intravitreal test articles. Key elements include: standardized slit lamp settings; expansion of criteria to enhance applicability to nonrabbit species; refinement and disambiguation of scoring criteria for corneal opacity, fluorescein staining severity, and aqueous flare; introduction of novel criteria for scoring of aqueous and anterior vitreous cell; and introduction of criteria for findings observed with drugs/devices targeting the posterior segment. A modified Standardization of Uveitis Nomenclature (SUN) system is also introduced to facilitate accurate use of SUN's criteria in laboratory species. The SPOTS systems provide criteria that stand to enhance the applicability of semiquantitative scoring criteria to the full range of laboratory species, in the context of modern approaches to ocular therapeutics and drug delivery and drug and device development.

Biomechanical, ultrastructural, and electrophysiological characterization of the non-human primate experimental glaucoma model

Laser-induced experimental glaucoma (ExGl) in non-human primates (NHPs) is a common animal model for ocular drug development. While many features of human hypertensive glaucoma are replicated in this model, structural and functional changes in the unlasered portions of trabecular meshwork (TM) of laser-treated primate eyes are understudied. We studied NHPs with ExGl of several years duration. As expected, ExGl eyes exhibited selective reductions of the retinal nerve fiber layer that correlate with electrophysiologic measures documenting a link between morphologic and elctrophysiologic endpoints. Softening of unlasered TM in ExGl eyes compared to untreated controls was observed. The degree of TM softening was consistent, regardless of pre-mortem clinical findings including severity of IOP elevation, retinal nerve fiber layer thinning, or electrodiagnostic findings. Importantly, this softening is contrary to TM stiffening reported in glaucomatous human eyes. Furthermore, microscopic analysis of unlasered TM from eyes with ExGl demonstrated TM thinning with collapse of Schlemm’s canal; and proteomic analysis confirmed downregulation of metabolic and structural proteins. These data demonstrate unexpected and compensatory changes involving the TM in the NHP model of ExGl. The data suggest that compensatory mechanisms exist in normal animals and respond to elevated IOP through softening of the meshwork to increase outflow.

Human retinal endothelial cells and astrocytes cultured on 3-D scaffolds for ocular drug discovery and development

Topical ocular ketorolac improves the outcomes of severe retinopathy of prematurity and when administered with systemic caffeine, decreases the severity of oxygen-induced retinopathy. We tested the hypothesis that co-cultures of human retinal endothelial cells (HRECs) and human retinal astrocytes (HRAs) on 3-dimensional (3-D) hydrogel scaffolds is a more representative biomimetic paradigm of the blood-retinal-barrier (BRB) than 2-D cultures, and should be utilized for preclinical drug discovery and development. Mono- and co-cultures of HRECs and HRAs were treated with standard doses of ketorolac, ibuprofen, and/or caffeine, and exposed to hyperoxia, intermittent hypoxia (IH), or normoxia on 2-D surfaces or 3-D biodegradable hydrogel scaffolds (AlgiMatrix or Geltrex). Media and cells were collected at 72h post treatment for arachidonic acid metabolites. Cells cultured on 3-D scaffolds exhibited less oxidative stress and variability in drug responses. HRAs enhanced the responses of HRECs to drugs and changes in oxygen environment. PGE2 and PGI2 were the predominant prostanoids produced in response to IH, reflecting COX-2 immunoreactivity. We conclude that HRECs and HRAs co-cultured on 3-D scaffolds may recapitulate drug responses of the dynamic BRB and therefore should be implemented for preclinical ocular drug discovery and development.

Imaging endpoints in clinical trials

SummaryClinical drug trials usually rely on primary endpoints that are well‐described direct measures of patient benefit or are closely related to clinical outcomes such as visual field testing. However, there is a lack of short‐term, easy to measure, highly reproducible endpoints predictive for visual acuity outcomes that would qualify as sufficiently predictive variables for patients′ benefit in clinical trials. This of special importance for the follow up of slowly developing diseases, which requires long‐term studies with large sample sizes. The rapid progression in the development of in‐vivo imaging techniques offers now new and exiting possibilities to assess human anatomic or physiologic information in unprecedented high resolution. Thus, the use of these new imaging techniques such as optical coherence tomography as possible endpoints in clinical phase III trials has been widely and controversially discussed. This talk aims to summarize the potential and limitations of modern imaging techniques to serve as surrogate parameters to substitute for clinical endpoints in ocular drug development.

General Pharmacokinetic Model for Topically Administered Ocular Drug Dosage Forms.

In ocular drug development, an early estimate of drug behavior before any in vivo experiments is important. The pharmacokinetics (PK) and bioavailability depend not only on active compound and excipients but also on physicochemical properties of the ocular drug formulation. We propose to utilize PK modelling to predict how drug and formulational properties affect drug bioavailability and pharmacokinetics. A physiologically relevant PK model based on the rabbit eye was built to simulate the effect of formulation and physicochemical properties on PK of pilocarpine solutions and fluorometholone suspensions. The model consists of four compartments: solid and dissolved drug in tear fluid, drug in corneal epithelium and aqueous humor. Parameter values and in vivo PK data in rabbits were taken from published literature. The model predicted the pilocarpine and fluorometholone concentrations in the corneal epithelium and aqueous humor with a reasonable accuracy for many different formulations. The model includes a graphical user interface that enables the user to modify parameters easily and thus simulate various formulations. The model is suitable for the development of ophthalmic formulations and the planning of bioequivalence studies.

HIF inhibitors for ischemic retinopathies and cancers: options beyond anti-VEGF therapies.

Aberrant activation of the hypoxia inducible factor (HIF) pathway causing overexpression of angiogenic genes, like vascular endothelial growth factor (VEGF), is one of the underlying causes of ocular neovascularization (NV) and metastatic cancer. Consistently, along with surgical interventions, a number of anti-VEGF agents have been approved by FDA for the treatment of ocular neovascular diseases. These anti-VEGF agents, like ranibizumab/lucentis, have revolutionized the treatment in the past decade. However, substantial vision improvement is observed only in a subset of age-related macular degeneration patients receiving ranibizumab. Further, all current therapies are associated with limitations and side effects. For example, surgeries cause tissue destruction and inflammation while anti-VEGF therapies are expensive, require repeated administration, and offer temporary relief from vascular leakage. These factors impose significant cost and treatment burdens to both the patient and society. With an aging population in most western countries with a continually increasing number of patients on lifelong treatment for these retinal diseases, the focus of ocular drug development for neovascular diseases will be to improve efficacy while reducing treatment costs. Blocking the HIF pathway, a major regulator of ocular NV and cancer, offers an appealing therapeutic strategy. Therefore, this review summarizes HIF inhibitors that have been recently evaluated for the treatment of different cancers and ischemic retinopathies.

Species Differences in the Geometry of the Anterior Segment Differentially Affect Anterior Chamber Cell Scoring Systems in Laboratory Animals

To determine the impact of anterior segment geometry on ocular scoring systems quantifying anterior chamber (AC) cells in humans and 7 common laboratory species. Using normative anterior segment dimensions and novel geometric formulae, ocular section volumes measured by 3 scoring systems; Standardization of Uveitis Nomenclature (SUN), Ocular Services On Demand (OSOD), and OSOD-modified SUN were calculated for each species, respectively. Calculated volumes were applied to each system's AC cell scoring scheme to determine comparative cell density (cells/mm(3)). Cell density values for all laboratory species were normalized to human values and conversion factors derived to create modified scoring schemes, facilitating interspecies comparison with each system, respectively. Differences in anterior segment geometry resulted in marked differences in optical section volume measured. Volumes were smaller in rodents than dogs and cats, but represented a comparatively larger percentage of AC volume. AC cell density (cells/mm(3)) varied between species. Using the SUN and OSOD-modified SUN systems, values in the pig, dog, and cat underestimated human values; values in rodents overestimated human values. Modified normalized scoring systems presented here account for species-related anterior segment geometry and facilitate both intra- and interspecies analysis, as well as translational comparison. Employment of modified AC cell scoring systems that account for species-specific differences in anterior segment anatomy would harmonize findings across species and may be more predictive for determining ocular toxicological consequences in ocular drug and device development programs.

Cytotoxicity of dimethyl sulphoxide on ocular cells in vitro

Background Dimethyl sulfoxide(DMSO) is a commonly used adjuvant to promote testing drug solubility to prepare multi-levels testing drug concentrations.DMSO is cell type-dependent cytotoxic and its toxicity can interfere the testing drug evaluation.Determining its safe concentration on commonly used cell types is important for ocular drug development. Objective This study was to determine the minimal toxic concentration of DMSO for in vitro ocular cell lines in a simulated drug screening setting. Methods Retinal pigment epithelial (RPE) cells were isolated from one pigmented rabbit and primarily cultured.Human RPE cell strain (ARPE19), scleral fibroblasts line (S75-Fron), human Muller cell line (MIO-M1), human lens epithelial cell line (HLEC), human choroidal melanoma cell line (OCM-1), human umbilical endothelial cell (HUVEC) and human HeLa cell line (HELA) were cultured.Different concentrations of DMSO (1.6%, 1.0%, 0.8%, 0.4%, 0.2% and 0.1%) were prepared with 160 μl DMSO solution and 9.84 ml RPMI1640 (or DMEM/F12 or DMEM) containing 2% fetal bovine serum.Different concentrations of DMSO were added in medium for 96 hours, and the and viability (absorbance) of the cells was detected using MTS to evaluate the cytotoxicity of DMSO. Results Rabbit primary RPE cells showed the yellow-green fluorescence for cytokeratin(CK) and HMB45 red fluorescence for S100.The viability of the cells was gradually declined as the increase of DMSO dose, showing significant differences in ARPE19, S75-Fron, HLEC, OCM-1, HUVEC and primary RPE cells (all at P 1.6%, >1.6%, 0.2%, 0.2%, 0.2%, respectively, and HUVEC was more sensitive to the cytotoxicity of DMSO (P=0.02), and MIO-M1 was the least sensitive to DMSO (P=0.39). The viability of HUVEC and primary RPE cells went down with the increase of DMSO dose, and S75-Fron viability started to decline in 0.1% DMSO and then stabilize with the higher concentrations until 1.6% DMSO at which the viability showed further decline. Conclusions The tolerability of ocular cells in vitro to DMSO varies depending on the cell types.The minimal toxic concentration ranged from 0.1% to 1.6%.The result suggests that a concurrent DMSO control should be set up along with the testing compound. Key words: Dimethyl sulfoxide; Cytotoxicity; Ocular cells; Drug screening

Topical drug delivery to retinal pigment epithelium with microfluidizer produced small liposomes

Drug delivery from topically instilled eye drops to the posterior segment of the eye has long been one of the greatest challenges of ocular drug development. We developed methods of liposome preparation utilizing a microfluidizer to achieve adjustable nanoparticle size (even less than 80nm) and high loading capacity of plasmid DNA. The microfluidizing process parameters were shown to affect the size of the liposomes. Higher operating pressures and passage for at least 10 times through the microfluidizer produced small liposomes with narrow size distribution. The liposomes were physically stable for several months at +4°C. In vivo distribution of the optimized liposome formulations in the rat eyes was investigated with confocal microscopy of the histological specimens. Transferrin was used as a targeting ligand directed to retinal pigment epithelium. Size dependent distribution of liposomes to different posterior segment tissues was seen. Liposomes with the diameter less than 80nm permeated to the retinal pigment epithelium whereas liposomes with the diameter of 100nm or more were distributed to the choroidal endothelium. Active targeting was shown to be necessary for liposome retention to the target tissue. In conclusion, these microfluidizer produced small liposomes in eye drops are an attractive option for drug delivery to the posterior segment tissues of the eye.