2-(2-Hydroxyethyl)piperazine derivatives as potent human carbonic anhydrase inhibitors: Synthesis, enzyme inhibition, computational studies and antiglaucoma activity.

Targeting Carbonic Anhydrases (CAs) represents a strategy to treat several diseases, from glaucoma to cancer. To widen the structure-activity relationships (SARs) of our series of piperazines endowed with potent human carbonic anhydrase (hCA) inhibition, a new series of chiral piperazines carrying a (2-hydroxyethyl) group was prepared. The Zn-binding function, the 4-sulfamoylbenzoyl moiety, was connected to one piperazine N-atom, while the other nitrogen was decorated with alkyl substituents. In analogy to the approach used for the synthesis of the previously reported series, the preparation of the new compounds started with (R)- and (S)-aspartic acid. A partial racemization occurred during the synthesis. In order to overcome this problem, other chemical strategies were investigated. The inhibitory activity of the new polar derivatives against four hCAs isoforms I, II, IV and IX using a stopped flow CO2 hydrase assay was determined. Some compounds showed potency in the nanomolar range and a preference for inhibiting hCA IX.

Drugs for the treatment of glaucoma: Targets, structure-activity relationships and clinical research.

Glaucoma is the third leading cause of blindness and impairment of vision worldwide, after refractive errors and cataracts. According to the survey, the number of people with glaucoma is more than 76 million, with projections increasing to 112 million by 2040. With the coming of an aging society, the number of people suffering from glaucoma will increase day by day. Glaucoma is a heterogeneous disease characterized by damage to the head of the optic nerve and visual field. High intraocular pressure is a major risk and cause of glaucoma optic neuropathy. Therefore, drug lowering intraocular pressure therapy is still the first-line therapy in clinical practice. Here, the targets, structure-activity relationship, and clinical progress of drugs for the treatment of glaucoma are reviewed.

Review of Approaches for Increasing Ophthalmic Bioavailability for Eye Drop Formulations.

Ophthalmic diseases represent a significant problem as over 2 billion people worldwide suffer from vison impairment and blindness. Eye drops account for around 90% of ophthalmic medications but are limited in success due to poor patient compliance and low bioavailability. Low bioavailability can be attributed to short retention times in the eye caused by rapid tear turnover and the difficulty of drug diffusion through the multi-layered structure of the eye that includes lipid-rich endothelial and epithelial layers as well as the stroma which is high in water content. In addition, there are barriers such as tight junctional complexes in the corneal epithelium, lacrimal turnover, nasolacrimal drainage, blinking reflexes, efflux transporters, drug metabolism by ocular enzymes, and drug binding to or repulsion from conjunctival mucins, tear proteins, and melanin. In order to maximize transport through the cornea while minimizing drug loss through other pathways, researchers have developed numerous methods to improve eye drop formulations including the addition of viscosity enhancers, permeability enhancers, mucoadhesives, and vasoconstrictors, or using formulations that include puncta occlusion, nanocarriers, or prodrugs. This review explains the mechanism behind each of these methods, examines their history, analyzes previous and current research, evaluates future applications, and discusses the pros and cons of each technique.

Gelling hypotonic polymer solution for extended topical drug delivery to the eye.

Eye-drop formulations should hold as high a concentration of soluble drug in contact with ocular epithelium for as long as possible. However, eye tears and frequent blinking limit drug retention on the ocular surface, and gelling drops typically form clumps that blur vision. Here, we describe a gelling hypotonic solution containing a low concentration of a thermosensitive triblock copolymer for extended ocular drug delivery. On topical application, the hypotonic formulation forms a highly uniform and clear thin layer that conforms to the ocular surface and resists clearance from blinking, increasing the intraocular absorption of hydrophilic and hydrophobic drugs and extending the drug-ocular-epithelium contact time with respect to conventional thermosensitive gelling formulations and commercial eye drops. We also show that the conformal gel layer allows for therapeutically relevant drug delivery to the posterior segment of the eyeball in pigs. Our findings highlight the importance of formulations that conform to the ocular surface before viscosity enhancement for increased and prolonged ocular surface contact and drug absorption.

Improved Ocular Tissue Models and Eye-On-A-Chip Technologies Will Facilitate Ophthalmic Drug Development.

In this study, we describe efforts by the National Eye Institute (NEI) and National Center for Advancing Translational Science (NCATS) to catalyze advances in 3-dimensional (3-D) ocular organoid and microphysiological systems (MPS). We reviewed the recent literature regarding ocular organoids and tissue chips. Animal models, 2-dimensional cell culture models, and postmortem human tissue samples provide the vision research community with insights critical to understanding pathophysiology and therapeutic development. The advent of induced pluripotent stem cell technologies provide researchers with enticing new approaches and tools that augment study in more traditional models to provide the scientific community with insights that have previously been impossible to obtain. Efforts by the National Institutes of Health (NIH) have already accelerated the pace of scientific discovery, and recent advances in ocular organoid and MPS modeling approaches have opened new avenues of investigation. In addition to more closely recapitulating the morphologies and physiological responses of in vivo human tissue, key breakthroughs have been made in the past year to resolve long-standing scientific questions regarding tissue development, molecular signaling, and pathophysiological mechanisms that promise to provide advances critical to therapeutic development and patient care. 3-D tissue culture modeling and MPS offer platforms for future high-throughput testing of therapeutic candidates and studies of gene interactions to improve models of complex genetic diseases with no well-defined etiology, such as age-related macular degeneration and Fuchs' dystrophy.

Understanding drug distribution and release in ophthalmic emulsions through quantitative evaluation of formulation-associated variables.

Establishing bioequivalence (BE) of ophthalmic emulsions in the absence of in vivo data is challenging. In these emulsions, drug release is a complex process due to drug distribution among various phases which are difficult to characterize. The objective of this study is to investigate the process of drug distribution and mechanism of drug release in the context of formulation-associated variables. A previously reported kinetic method for determining drug partitioning was used to quantitatively evaluate the drug distribution within a simplified biphasic (emulsion) system employing cyclosporine and difluprednate as model drugs. The impacts of formulation variables, such as the amount of polysorbate 80, glycerin, and carbomer copolymer as well as the area of oil-water interface were investigated. Polysorbate 80 was found to have the greatest influence on the drug distribution. It enhanced both the rate and extent of the drug distribution from oil to aqueous phase. Glycerin was found to slightly reduce the rate and extent of drug distribution of cyclosporine into the aqueous phase, probably by suppressing the solubilization capability of the micelles. Carbomer slowed down the diffusion of drug into the oil phase and shifted the equilibrium drug distribution towards the aqueous phase. Furthermore, increase in the interfacial area significantly increased the rate of drug diffusion across the oil-aqueous interface but had negligible effect on the extent of drug distribution. It is noteworthy that the experimental setup utilized a planar interface rather than an interface with curvature, which may have slightly underestimated the influence of globule size on equilibrium drug distribution. The findings of this study give insight into the drug distribution and diffusion in complex ophthalmic emulsions and assist with formulation design as well as development of in vitro methods to support BE assessment of ophthalmic emulsions.

Levofloxacin Hemihydrate In Situ Gelling Ophthalmic Solution: Formulation Optimization and In Vitro and In Vivo Evaluation.

Bacterial conjunctivitis is a leading cause of ocular infections requiring short-term therapeutic treatment with frequent administration of drugs on daily basis. Topical dosage forms available in the market for the treatment of bacterial conjunctivitis such as simple drug solutions and suspensions are rapidly eliminated from the precorneal space upon instillation due to tear turn over and nasolacrimal drainage, limiting intraocular bioavailability of drug to less than 10% of the administered dose. To overcome issues related to conventional drop, an effort was made to design and evaluate prolong release ophthalmic solution of levofloxacin hemihydrate (LFH) using ion-sensitive in situ gelling polymer. Gellan gum was used as the in situ gelling agent. Formulations were screened based on in vitro gelation time, in vitro drug release, and stability towards sol to gel conversion upon storage. The prototype formulations exhibiting quick in vitro gelling time (< 15 s), prolonged in vitro drug release (18-24 h), and stability for at least 6 months at 25°C/40% relative humidity (RH) and 40°C/25% RH were evaluated for pharmacokinetic studies using healthy New Zealand white rabbits. Tested formulations were found to be well-tolerated and showed significant increase in AUC0-24 (22,660.39 h ng/mL) and mean residence time (MRT 12 h) as compared with commercially available solution Levotop PF® (Ajanta Pharma Ltd., India)(AUC0-24 6414.63 h ng/mL and MRT 4 h). Thus, solution formulations containing in situ gelling polymer may serve as improved drug delivery system providing superior therapeutic efficacy and better patient compliance for the treatment of bacterial conjunctivitis.

Sustained release of ketorolac tromethamine from poloxamer 407/cellulose nanofibrils graft nanocollagen based ophthalmic formulations.

There has been extensive utilization of poloxamer 407 (PM) for the delivery of various ophthalmic drugs aimed at efficient ophthalmic drug delivery approach for longer precorneal residence time along with acceptable bioavailability of drugs. We have studied the effect of nanocellulose grafted collagen (CGC) on the performance of in situ gels based on PM for the controlled in vitro release of Ketorolac Tromethamine (KT). CGC has shown great influence evident by the reduction in PM critical gelation concentration, increased gel strength, and prolonged the release of loaded drugs compared with the virgin PM gel. The engineered nanocomposite formulations established an anomalous diffusion mechanism along with a Fickian diffusion controlled drug release for 1.5 & 1.75 w/v% CGC reinforced PM. Hence, the synthesized in situ nanocomposites are potential candidates for ophthalmic drug delivery system.

Designing of a pH-Triggered Carbopol®/HPMC In Situ Gel for Ocular Delivery of Dorzolamide HCl: In Vitro, In Vivo, and Ex Vivo Evaluation.

Dorzolamide HCl (DRZ) ophthalmic drop is one of the most common glaucoma medications which rapidly eliminates after instillation leading to short residence time of the drug on cornea. The purpose of the present study is to develop a pH-triggered in situ gel system for ophthalmic delivery of DRZ for treatment of ocular hypertension. In this study, a 32 full factorial design was used for preparation of in situ gel formulations using different levels of Carbopol® and hydroxyl propyl methyl cellulose (HPMC). Rheological behavior, in vitro drug release, ex vivo corneal permeability, and IOP-lowering activity were investigated. DRZ solution (2% w/v) containing of 0.1% (w/v) Carbopol® and 0.1% (w/v) HPMC was selected as the optimal formulation considering its free flow under non-physiological conditions (initial pH and 25 ± 2°C) and transition to appropriate gel form under physiological circumstance (pH 7.4 and 34°C). This in situ gel presented the mucoadhesive property. Ex vivo corneal permeability of this combined solution was similar to those of DRZ solution. The developed formulation compared to the marketed drop (Biosopt®) and DRZ 2% solution had a better performance in intraocular pressure activity. The efficiency and long duration of IOP reduction could be due to the prolonged residence time of the in situ gel. The presence of Carbopol® as a pH triggered and mucoadhesive polymer causes to attach to the ocular mucosal surface for a long term.

Analog Derivatization of Cannabidiol for Improved Ocular Permeation.

Purpose: Cannabidiol (CBD), active component of plant Cannabis sativa, has anti-inflammatory properties that could potentially help treat diabetic retinopathy-induced pain and inflammation. However, CBD is a lipophilic molecule making its topical delivery to back of the eye challenging. This study aims at improving ocular penetration of CBD by means of analog derivatization. Methods: Analogs were designed using various ligands, such as amino acids (AAs) and dicarboxylic acids (DCAs) and their combinations. Select analogs were screened in vitro with respect to their stability in ocular tissue homogenates. Based on in vitro stability, analogs were selected for in rabbits testing. Formulations containing these compounds were tested in rabbits to determine ocular tissue disposition of CBD and the analogs after topical application. The rabbits were sacrificed 90 min post-topical application and the aqueous humor, vitreous humor (VH), iris-ciliary bodies (IC), and retina-choroid (RC) were analyzed for CBD and analog content. Results: CBD-divalinate-dihemisuccinate (CBD-Di-VHS) and CBD-divalinate (CBD-Di-Val) were stable in the ocular tissue homogenates. Post-topical application, CBD and CBD-Di-Val analog levels were detected only in RC. Dosing of CBD-Di-VHS nanoemulsion generated analog levels both in the VH and in the RC, respectively. In contrast, post dosing of CBD-monovalinate-monohemisuccinate (CBD-Mono-VHS), both the analog and CBD were detected in the IC and RC. Conclusion: The analogs demonstrated superior penetration into ocular tissues in comparison with CBD. CBD-Di-VHS and CBD-Mono-VHS exhibited better permeation properties, possibly due to improved stability and physicochemical characteristics imparted by AA and DCA combination derivatives.

Synthetic Approaches to the New Drugs Approved During 2017.

New drugs introduced to the market every year represent privileged structures for particular biological targets. These new chemical entities (NCEs) provide insight into molecular recognition while serving as leads for designing future new drugs. This annual review describes the most likely process-scale synthetic approaches to 31 new chemical entities approved for the first time globally in 2017.

Ocular Ciliopathies: Genetic and Mechanistic Insights into Developing Therapies.

Developing suitable medicines for genetic diseases requires a detailed understanding of not only the pathways that cause the disease, but also the identification of the genetic components involved in disease manifestation. This article focuses on the complexities associated with ocular ciliopathies - a class of debilitating disorders of the eye caused by ciliary dysfunction. Ciliated cell types have been identified in both the anterior and posterior segments of the eye. Photoreceptors (rods and cones) are the most studied ciliated neurons in the retina, which is located in the posterior eye. The photoreceptors contain a specialized lightsensing outer segment, or cilium. Any defects in the development or maintenance of the outer segment can result in severe retinal ciliopathies, such as retinitis pigmentosa and Leber congenital amaurosis. A role of cilia in the cell types involved in regulating aqueous fluid outflow in the anterior segment of the eye has also been recognized. Defects in these cell types are frequently associated with some forms of glaucoma. Here, we will discuss the significance of understanding the genetic heterogeneity and the pathogenesis of ocular ciliopathies to develop suitable treatment strategies for these blinding disorders.

Consensus SEO-SEFH of recommendations for use and compounding of ophthalmic preparations.

There are many medicinal products that, although having shown efficacy and  safety in different ophthalmological indications, they are not authorized or  commercially available for ophthalmic administration. This implies, on one hand,  that they must be used according to legislation that regulates the availability of  medicines in special situations and, on the other hand, that they must be  prepared in the pharmacy services for ophthalmic administration, according to  quality criteria to ensure its effectiveness, stability and sterility. This document  gathers the consensus between the Spanish Society of Ophthalmology and the  Spanish Society of Hospital Pharmacy about these selected preparations which  have shown enough evidence in their efficacy and safety for their ophthalmic use (off label) and ophthalmic administration. This document includes  recommendations about its use according to the current legislation. In addition,  with the aim of harmonizing the preparation of intraocular injections in the  hospital pharmacy services, general recommendations are set in this document  to ensure the compliance with standards established in the Spanish Guideline for Good Preparation Practices of Medicinal Products in Hospital Pharmacies. These  recommendations include sections such as the area of preparation, material,  technique, packaging, stability, quality control, prescription and traceability of  intraocular preparations.

Son muchos los medicamentos que, aun habiendo demostrado eficacia y seguridad en diferentes indicaciones oftalmológicas, no están autorizados ni disponibles comercialmente en una forma adecuada para esta vía de  administración.Esto implica, por un lado, que se deban utilizar según la legislación que regula la disponibilidad de medicamentos en situaciones especiales y, por otro, que se  deban preparar en los Servicios de Farmacia para su administración por vía  oftálmica, conforme a unos criterios de calidad que aseguren su efectividad,  estabilidad y esterilidad. Este documento recoge un consenso entre la Sociedad  Española de Oftalmología y la Sociedad Española de Farmacia Hospitalaria sobre  aquellas preparaciones con suficiente evidencia respecto a su eficacia y  seguridad para su uso no autorizado en indicaciones y vía de administración  oftálmicas. Se incluyen recomendaciones para su utilización de acuerdo con la  legislación vigente. Además, con el ánimo de armonizar la preparación de  inyecciones intraoculares en los Servicios de Farmacia Hospitalaria, se  establecen unas recomendaciones generales para su elaboración siguiendo los  estándares establecidos en la Guía de Buenas Prácticas de Preparación de  Medicamentos en los Servicios de Farmacia Hospitalaria. En estas  recomendaciones se incluyen apartados como el lugar de preparación, el  material, la técnica, el envasado, el periodo de validez, el control de calidad, la  prescripción y la trazabilidad de las preparaciones intraoculares.

Development of an Aqueous Ophthalmic Solution with an Enhanced-Solubility Enrofloxacin Crystal, and its Clinical Evaluation in Dogs.

BACKGROUND: The concern about the frequent use of ciprofloxacin in veterinary medicine is linked to increased antimicrobial resistance. The corresponding fluoroquinolone for veterinary use is enrofloxacin. A new solvate form of enrofloxacin, as dihydrate-hydrochloride (enro-C) with higher water solubility than the parent compound, was formulated as an ophthalmic solution (pH 5). A multicentre, longitudinal, non-inferiority clinical study in a non-hospital environment was designed to treat 36 dogs affected by tobramycin-unresponsive conjunctivitis with either the experimental 0.5% enro-C ophthalmic preparation (enro-CG) or a commercial preparation of ciprofloxacin (cipro-G). Other causes of conjunctivitis were ruled out. Pathogens were isolated and minimum inhibitory concentration (MIC) studies of tobramycin were carried out. Three blocks of bacterial resistance were set up, beginning at the established breakpoint i.e., 4 µg/mL; 8 µg/mL and 16 µg/mL. Eighteen dogs were randomly assigned to each block. The enro-CG group was treated with two drops of the referred preparation (10 mg/eye) twice a day for 7 days, and the cipro-G group was treated with four drops of a 0.3% commercially available ciprofloxacin eye-drop preparation (9 mg/eye) twice a day, also for 7 days. Clinical and bacteriological cure rates were evaluated. RESULTS: Enro-C-treated dogs achieved a clinical cure one day earlier than ciprofloxacin-treated dogs, and unlike this latter group, enro-CG achieved bacteriological cure in all cases. No side effects were observed in either group, but dogs treated with enro-C showed no discomfort, allowing easier treatment-compliance. CONCLUSION: This is the first study reported on the successful formulation of enrofloxacin as an ophthalmic solution. Clinical assessment reveals outstanding clinical efficacy. It is necessary to conduct further research on clinical efficacy and toxicity, with the chronic use of this preparation under different clinical challenges.

Octa-arginine modified lipid emulsions as a potential ocular delivery system for disulfiram: A study of the corneal permeation, transcorneal mechanism and anti-cataract effect.

The purpose of the study was to design a novel octa-arginine (R8) modified lipid emulsion (LE) system for the ocular delivery of the lipophilic drug disulfiram (DSF). The influence of the particle size of the lipid emulsions and the presence of R8 on corneal permeation was studied. DSF-loaded lipid emulsions with different particle sizes (DSF-LE1, DSF-LE2, DSF-LE3) and DSF-loaded lipid emulsions modified with R8 (DSF-LE1-R8 and DSF-LE2-R8) were prepared. The Zeta potential of the lipid emulsions was changed from negative to a positive value after modification of R8. The mucoadhesion of different preparations was investigated, and DSF-LE1-R8 was found to produce the strongest mucoadhesion. The in vitro corneal penetration study and in vivo ocular distribution study showed that the R8 modified lipid emulsion (DSF-LE1-R8) with a nano particle size, exhibited the highest permeability and the largest amount of DDC distributed in ocular issues. Coumarin-6 labelled LE1-R8 displayed more homogeneous fluorescence with the deeper penetration into the cornea compared with other preparations at various times. Confocal laser scanning microscopy showed that, in addition to paracellular routes, LE-R8 could also transport across the corneal epithelium by transcellular routes as a result of increased uptake due to the R8 modification. Furthermore, the anti-cataract effect was evaluated and it was found that DSF-LE1-R8 exhibited a marked anti-cataract effect. Therefore, the lipid emulsions with nano-sized particles and modification of R8 were proposed as a potential ocular delivery system to improve the corneal penetration and ocular delivery of DSF.

Ocular Cubosome Drug Delivery System for Timolol Maleate: Preparation, Characterization, Cytotoxicity, Ex Vivo, and In Vivo Evaluation.

Glaucoma is an ocular disease featuring increased intraocular pressure (IOP) and its primary treatment strategy is to lower IOP by medication. Current ocular drug delivery in treating glaucoma is confronting a variety of challenges, such as low corneal permeability and bioavailability due to the unique anatomical structure of the human eye. To tackle these challenges, a cubosome drug delivery system for glaucoma treatment was constructed for timolol maleate (TM) in this study. The TM cubosomes (liquid crystalline nanoparticles) were prepared using glycerol monooleate and poloxamer 407 via high-pressure homogenization. These constructed nanoparticles appeared spherical using transmission electron microscopy and had an average particle size of 142 nm, zeta potential of -6.27 mV, and over 85% encapsulation efficiency. Moreover, using polarized light microscopy and small-angle X-ray scattering (SAXS), it was shown that the TM cubosomes have cubic liquid crystalline D-type (Pn3m) structure, which provides good physicochemical stability and high encapsulation efficiency. Ex vivo corneal permeability experiments showed that the total amount of TM cubosomes penetrated was higher than the commercially available eye drops. In addition, in vivo studies revealed that TM cubosomes reduced the IOP in rabbits from 27.8∼39.7 to 21.4∼32.6 mmHg after 1-week administration and had a longer retention time and better lower-IOP effect than the commercial TM eye drops. Furthermore, neither cytotoxicity nor histological impairment in the rabbit corneas was observed. This study suggests that cubosomes are capable of increasing the corneal permeability and bioavailability of TM and have great potential for ocular disease treatment.

Preparation and evaluation of HPMC-based pirfenidone solution in vivo.

CONTEXT: Pirfenidone (PFD) has exhibited therapeutic potential in the treatment of cell proliferative disorders. The previously developed 0.5% water-based PFD eye drops by our team exhibited antiscarring effectiveness and ocular safety but with a limit of short half-life and poor bioavailability. OBJECTIVE: To increase bioavailability of the water-based PFD eye drops, we prepared a viscous solution by adding hydroxypropyl methylcellulose (HPMC, F4M), which acted as a viscosity-enhancer. Subsequently, we compared the HPMC-based PFD solution with the water-based PFD eye drops. MATERIALS AND METHODS: PFD solution with 1% HPMC (w/v) was prepared, and the viscosities at different shear rates were measured to investigate its rheology. PFD concentrations in the tear, aqueous humor, conjunctiva, cornea, and sclerae of New Zealand rabbits were detected at different time points with high-performance liquid chromatography (HPLC) following single instillation of the 0.5% PFD (w/v) water-based eye drops or HPMC-based solution. RESULTS: Compared with the 0.5% water-based PFD eye drops, the HPMC-based solution increased the PFD levels in tears and prolonged the residence time from 10 to more than 20 min (p < .01). Consequently, the concentrations of PFD in aqueous humor, conjunctiva, cornea, and sclera were elevated to varying degrees until 90 min after topical administration. CONCLUSIONS: The developed formulation possesses a same readily administration and simple preparation as the PFD eye drops; however, the HPMC-based solution exhibited the higher bioavailability.

Preparation and evaluation of a timolol maleate drug-resin ophthalmic suspension as a sustained-release formulation in vitro and in vivo.

The aim of this work was to assess the performance of resin as an ocular delivery system. Timolol maleate (TM) was chosen as the model drug and an ion exchange resin (IER) as the carrier. The drug-resin complex was prepared using an oscillation method and then characterized regarding particle size, zeta potential, morphology, and drug content. After in vitro drug release study and corneal permeation study were performed, in vivo studies were performed in New Zealand albino rabbits using a suspension with particles sized 4.8 ± 1.2 µm and drug loading at 43.00 ± 0.09%. The results indicate that drug released from the drug-resin ophthalmic suspension permeated the cornea and displayed a sustained-release behavior. Drug levels in the ocular tissues after administration of the drug-resin ophthalmic suspension were significantly higher than after treatment with an eye drop formulation but were lower in body tissues and in the plasma. In conclusion, resins have great potential as effective ocular drug delivery carriers to increase ocular bioavailability of timolol while simultaneously reducing systemic drug absorption.

Delivery of Topically Applied Calpain Inhibitory Peptide to the Posterior Segment of the Rat Eye.

We developed an inhibitory peptide that specifically acts against mitochondrial µ-calpain (Tat-µCL, 23 amino acid, 2857.37 Da) and protects photoreceptors in retinal dystrophic rats. In the present study, we topically administered Tat-µCL to the eyes of Sprague-Dawley rats for 7 days to determine both the delivery route of the peptide to the posterior segment of the eye and the kinetics after topical application in adult rats. Distribution of the peptide was determined by immunohistochemical analysis, and enzyme-linked immune-absorbent assay was used to quantify the accumulation in the retina. Peptides were prominently detected in both the anterior and posterior segments of the eye at 1 h after the final eye drop application. Immunohistochemically positive reactions were observed in the retina, optic nerve, choroid, sclera and the retrobulbar tissues, even in the posterior portion of the eye. Immunoactivities gradually diminished at 3 and 6 h after the final eye drop. Quantitative estimations of the amount of peptide in the retina were 15.3, 5.8 and 1.0 pg/µg protein at 1, 3 and 6 h after the final instillation, respectively. Current results suggest that while the topically applied Tat-µCL peptide reaches the posterior segment of the retina and the optic nerve, the sufficient concentration (> IC50) is maintained for at least 6 h in the rat retina. Our findings suggest that delivery of topically applied peptide to the posterior segment and optic nerve occurs through the conjunctiva, periocular connective tissue, sclera and optic nerve sheath.