Subconjunctival Administration Research Articles

Pharmacokinetics of Bevacizumab after Topical, Subconjunctival, and Intravitreal Administration in Rabbits

To investigate the pharmacokinetics of bevacizumab in rabbits for three different routes of administrations: intravitreal injection, subconjunctival injection, and eye drops. Pigmented rabbits received bevacizumab in one eye by topical eye drops (1.25 mg/0.05 mL six times daily for the first 7 days), single subconjunctival injection (1.25 mg/0.05 mL), or single intravitreal injection (1.25 mg/0.05 mL). Bevacizumab concentrations in plasma and ocular tissues in the treated and fellow eyes were determined by sandwich enzyme-linked immunosorbent assay at 1, 2, 4, and 12 weeks after administration. After intravitreal injection in the treated eye, the mean maximum concentrations (C(max)) of bevacizumab in the iris/ciliary body and retina/choroid were 109,192.6, and 93,990.0 ng/g, respectively, whereas after subconjunctival injection, the C(max) was 1418.7 and 295.8 ng/g, respectively. In the fellow eyes, when the drug was administered by intravitreal injection, the C(max) was 753.6 ng/g in the iris/ciliary body and 224.2 ng/g in the retina/choroid and by subconjunctival injection was 1192.9 and 187.0 ng/g, respectively. With eye drops, only a small level of bevacizumab was detected in the iris/ciliary body and retina/choroid. Systemic exposure to bevacizumab was at the same level when administered by intravitreal or subconjunctival injection. Intravitreal injection of bevacizumab was the most effective route of administration for intraocular tissue. Also, bevacizumab injected subconjunctivally was transported into the intraocular tissues of the treated eyes at an effective level. Both intravitreal and subconjunctival injections of bevacizumab resulted in high plasma concentrations. Bevacizumab was distributed into the intraocular tissues in fellow eyes via the systemic circulation. This treatment may be effective for blocking vascular endothelial growth factor activity.

Prevention and Treatment of Corneal Neovascularization: Comparison of Different Doses of Subconjunctival Bevacizumab with Corticosteroid in Experimental Rats

Background: To evaluate and compare the effect of different doses of subconjunctival bevacizumab with betamethasone on the development of corneal major new vessels in a rat model of corneal chemical injury. Methods: The right eyes of 100 male Sprague-Dawley rats were randomly divided into 10 experimental groups (n = 10 per group). Chemical cauterization of the cornea was performed by using silver nitrate/potassium nitrate sticks. Immediately following corneal cauterization, the animals in groups 1–5 received subconjunctival injections of 0.02 ml of normal saline (control A), betamethasone LA (6 mg/ml) and different doses of bevacizumab (1, 5 and 25 mg/ml), respectively. In another experiment, the animals in groups 6–10 received subconjunctival injections of 0.02 ml of normal saline (control B), betamethasone LA (6 mg/ml) and different doses of bevacizumab (1, 5 and 25 mg/ml), respectively, 7 days following corneal cauterization. The numbers of major thick-walled vessels originating from the limbus reaching the corneal scar were counted 7 days after corneal cauterization in groups 1–5 and 14 days after corneal cauterization in groups 6–10. Results: The number of major vessels in groups 1–5 was 19.63 ± 3.77, 17.25 ± 5.33, 16.10 ± 5.02, 12.89 ± 2.70 and 12.36 ± 4.45 when assessed 7 days after corneal cauterization, respectively. Administration of betamethasone in group 2 had no significant effect on the corneal major vessel count compared to control A. The number of major vessels in groups 4 and 5 (bevacizumab 5 and 25 mg/ml) was significantly lower than that of group 1 (p < 0.01, Student’s t test). The number of vessels in groups 6–10 was 12.55 ± 5.64, 11.30 ± 9.33, 5.50 ± 6.34, 2.73 ± 4.73 and 2.67 ± 3.77 when assessed 14 days after corneal cauterization, respectively. Subconjunctival administration of betamethasone 7 days after corneal cauterization did not reduce the amount of corneal major vessels compared to control B. Administration of 0.02 ml of bevacizumab in doses of 1, 5 and 25 mg/ml 7 days after corneal cauterization significantly reduced the amount of major vessels compared to group 6 (p = 0.01, p < 0.01 and p < 0.01, respectively). There was no significant difference in percent area of corneal scar between different groups. Conclusion: Single subconjunctival injection of bevacizumab is efficacious in the prevention of formation as well as regression of major vessels compared to betamethasone in this rat model of corneal neovascularization. Even lower doses of bevacizumab might be efficacious.

Sustained delivery of timolol maleate from poly(lactic-co-glycolic acid)/poly(lactic acid) microspheres for over 3 months

It is estimated that 2.2 million people have glaucoma in the US and 67 million people worldwide. The majority of cases are associated with elevated intraocular pressure (IOP) and decreasing IOP eliminates or greatly reduces degeneration in most cases, including cases in which the IOP is in the normal range but optic neuropathy occurs. Timolol maleate has the longest record of safety and efficacy to lower IOP and is administered via eye drops one or more times per day. Unfortunately, compliance is poor across patient populations leading to degeneration. Patients typically see their ophthalmologist once every 3–4 months. If one could administer a long-acting treatment while in the doctor's office, one might overcome the compliance issue and effectively preserve sight. The critical step is to develop a formulation for timolol maleate that leads to sustained delivery for greater than 90 days and would permit a different treatment paradigm, namely subconjunctival administration once every 3–4 months. By using a 50 : 50 blend of PLGA 502H and PLA, this study was able to fabricate microspheres that delivered timolol maleate continually over 107 days, well within the time frame needed to make subconjunctival administration feasible and permit a new approach to treating glaucoma and diseases of the eye more broadly.

Modeling of Corneal and Retinal Pharmacokinetics after Periocular Drug Administration

To develop pharmacokinetics models to describe the disposition of small lipophilic molecules in the cornea and retina after periocular (subconjunctival or posterior subconjunctival) administration. Compartmental pharmacokinetics analysis was performed on the corneal and retinal data obtained after periocular administration of 3 mg of celecoxib (a selective COX-2 inhibitor) to Brown Norway (BN) rats. Berkeley Madonna, a differential and difference equation-based modeling software, was used for the pharmacokinetics modeling. The data were fit to different compartment models with first-order input and disposition, and the best fit was selected on the basis of coefficient of regression and Akaike information criteria (AIC). The models were validated by using the celecoxib data from a prior study in Sprague-Dawley (SD) rats. The corneal model was also fit to the corneal data for prednisolone at a dose of 2.61 mg in albino rabbits, and the model was validated at two other doses of prednisolone (0.261 and 26.1 mg) in these rabbits. Model simulations were performed with the finalized model to understand the effect of formulation on corneal and retinal pharmacokinetics after periocular administration. Celecoxib kinetics in the BN rat cornea can be described by a two-compartment (periocular space and cornea, with a dissolution step for periocular formulation) model, with parallel elimination from the cornea and the periocular space. The inclusion of a distribution compartment or a dissolution step for celecoxib suspension did not lead to an overall improvement in the corneal data fit compared with the two-compartment model. The more important parameter for enhanced fit and explaining the apparent lack of an increase phase in the corneal levels is the inclusion of the initial leak-back of the dose from the periocular space into the precorneal area. The predicted celecoxib concentrations from this model also showed very good correlation (r = 0.99) with the observed values in the SD rat corneas. Similar pharmacokinetics models explain drug delivery to the cornea in rat and rabbit animal models. Retinal pharmacokinetics after periocular drug administration can be explained with a four-compartment (periocular space, choroid-containing transfer compartment, retina, and distribution compartment) model with elimination from the periocular space, retina, and choroid compartment. Inclusion of a dissolution-release step before the drug is available for absorption or elimination better explains retinal t(max). Good fits were obtained in both the BN (r = 0.99) and SD (r = 0.99) rats for retinal celecoxib using the same model; however, the parameter estimates differed. Corneal and retinal pharmacokinetics of small lipophilic molecules after periocular administration can be described by compartment models. The modeling analysis shows that (1) leak-back from the site of administration most likely contributes to the apparent lack of an increase phase in corneal concentrations; (2) elimination via the conjunctival or periocular blood and lymphatic systems contributes significantly to drug clearance after periocular injection; (3) corneal pharmacokinetics of small lipophilic molecules can be explained by using similar models in rats and rabbits; and (4) although there are differences in some retinal pharmacokinetics parameters between the pigmented and nonpigmented rats, the physiological basis of these differences has yet to be ascertained.

Targeting MDM2 and MDMX in Retinoblastoma

Retinoblastoma is the third most common form of cancer in infants, and metastatic retinoblastoma is lethal in approximately 90% of cases. Early detection and aggressive therapy has resulted in a 95% probability of survival for retinoblastoma patients in the United States. However, the United States only represents 3-4% of the retinoblastoma cases worldwide. The majority of children diagnosed with retinoblastoma each year live in developing countries where the probability of survival is closer to 50%. This difference in survival rates reflects poor early detection rates and limited resources for the aggressive therapy necessary to treat retinoblastoma and manage the side effects associated with broad-spectrum systemic chemotherapy in young children. In order to have the most significant impact on retinoblastoma treatment in the United States and worldwide, current efforts have focused on local delivery of targeted chemotherapy. In this review, we summarize recent data showing that the p53 pathway is inactivated in 75% of retinoblastoma patients due to extra copies of the MDM2 and MDMX genes. A small molecule inhibitor of MDM2 called nutlin-3 can induce p53-mediated cell death in retinoblastoma cells. Subconjunctival delivery of nutlin-3 in preclinical models of retinoblastoma confirmed the efficacy of this approach in vivo. The advantage of local application of targeted chemotherapeutic agents such as nutlin-3 is that greater intraocular drug concentrations can be achieved without the side effects associated with systemic broad-spectrum chemotherapy. We propose that subconjunctival administration of targeted chemotherapy may be the best treatment option for children with retinoblastoma in the United States and throughout the developing world because it provides greater tumor response without the costs and complications associated with current treatment protocols.

Severe pseudo–preseptal cellulitis following sub–Tenon’s carboplatin injection for intraocular retinoblastoma

Sub-Tenon or subconjunctival administration of carboplatin is used to consolidate tumor regression by achieving high intraocular concentrations, without incurring systemic toxicity, in selected patients with intraocular retinoblastoma. Mild transient periocular edema is a common side effect of this treatment. We describe four patients with severe local soft-tissue toxic reaction mimicking preseptal cellulitis following subtenon carboplatin injections. The development of this complication after injecting through the inferior conjunctiva and unexpectedly severe signs are the unusual features of these patients. We did not encounter this complication after injections through the superior conjunctiva in 22 other patients using the same technique and identical drug dosage.

Posterior Juxtascleral Depot Administration of Anecortave Acetate

Objective To develop a safe and effective transcleral delivery of anecortave acetate, a novel angiostatic cortisene, in therapeutic concentrations to the choroid and retina in the region of the macula in patients with age related macular degeneration. Methods In pre-clinical studies with rabbits and monkeys, several routes of delivery were studied including oral and other systemic routes as well as topical, subconjunctival, intravitreal injections and posterior juxtascleral administration. In addition, posterior juxtascleral depot administration using a specially designed blunt cannula was evaluated in humans. Results Oral and other systemic routes were not acceptable due to rapid systemic metabolism. Topical and subconjunctival administrations resulted in subtherapeutic concentrations in the macular region (<0.1 μm). Intravitreal injections resulted in adequate drug levels, but the visual axis was obscured due to the opaque nature of the drug, and this method carries risks of endophthalmitis and retinal detachment. Posterior juxtascleral depot administration in rabbits and monkeys resulted in adequate retinal and choroidal drug levels (≥0.1 μm) up to 6 months after administration. In clinical studies, this administration technique was found to be safe. Conclusions Posterior juxtascleral depot administration of anecortave acetate onto the scleral surface near the macula is a safe and effective method for delivering therapeutic concentrations of drug to the macular region of the choroid and retina for up to 6 months.

Keeping the Cornea Clear

Although parts of the eye are highly vascularized, the ability to see clearly depends on the transparency--and thus the avascularity--of the cornea. Inflammation can promote corneal vascularization. Indeed, trachoma and herpetic keratitis, respectively the leading causes of blindness worldwide and in industrialized nations, are both associated with corneal hemangiogenesis (CHA). A group that previously showed that intact corneal epithelium (the outer layer of the cornea) suppresses CHA investigated the possibility that vascular endothelial growth factor receptor 3 (VEGFR3), which they had found in human corneal epithelial cells, might inhibit inflammatory CHA. Cursiefen et al. used a combination of immunohistochemistry, fluorescence-activated cell sorting (FACS) analysis, and reverse transcription polymerase chain reaction (RT-PCR) to confirm the presence of VEGFR3 on murine corneal epithelial cells (MCEs); further, VEGF-C bound to VEGFR3 on cultured MCEs and stimulated its phosphorylation. After demonstrating that corneal epithelium inhibited CHA following stimuli that elicited an inflammatory response in mouse eyes (corneal cauterization or placement of sutures in the corneal stroma), the authors showed that corneal suturing promoted up-regulation of mRNA encoding the VEGFR3 ligands VEGF-C and VEGF-D. Subconjunctival administration to cauterized de-epithelialized eyes of a chimeric protein containing the mouse VEGFR3 extracellular domain inhibited CHA, whereas the antiangiogenic capacity of corneal epithelium was inhibited by a blocking antibody directed against VEGFR3. Thus, the authors propose that the ectopic expression of VEGFR3 on corneal epithelial cells may act as a trap to keep vascularizing growth factors away from target cells in vascular endothelium. C. Cursiefen, L. Chen, M. Saint-Geniez, P. Hamrah, Y. Jin, S. Rashid, B. Pytowski, K. Persaud, Y. Wu, J. W. Streilein, R. Dana, Nonvascular VEGF receptor 3 expression by corneal epithelium maintains avascularity and vision. Proc. Natl. Acad. Sci. U.S.A. 103 , 11405-11410 (2006). [Abstract] [Full Text]

An outbreak of iritis and uveitis in dairy cattle at pasture associated with the supplementary feeding of baleage

Abstract Ocular disease associated with infection by Listeria monocytogenes has been reported in the veterinary literature for over 30 years (Kummeneje and Mikkelsen 1975), however it remains a poorly-defined condition (Morin 2004). Ocular listeriosis has generally been reported as having a similar disease pattern to that associated with listerial abortion or meningoencephalitis, i.e. the disease is primarily sporadic in occurrence although outbreaks can occur (Morgan 1977; Bocklisch et al 1991; Khodakaram-Tafti and Ikede 2005). However, Watson (1989) reported an outbreak of iritis and anterior uveitis in housed cattle linked to ocular infection with L. monocytogenes and associated with the feeding of silage. Since then, bovine iritis, commonly referred to as ‘silage eye’, has been increasingly recognised as a syndrome in dairy cattle in the United Kingdom (UK). Indeed, outbreaks of iritis are now more commonly reported by veterinarians in the UK than infectious bovine keratoconjunctivitis (Watson 2004), ...

Sustained release of nanosized complexes of polyethylenimine and anti-TGF-β2 oligonucleotide improves the outcome of glaucoma surgery

The purpose of this study was to design microspheres combining sustained delivery and enhanced intracellular penetration for ocular administration of antisense oligonucleotides. Nanosized complexes of antisense TGF-β2 phosphorothioate oligonucleotides (PS-ODN) with polyethylenimine (PEI), and naked PS-ODN were encapsulated into poly(lactide-co-glycolide) microspheres prepared by the double-emulsion solvent evaporation method. The PS-ODN was introduced either naked or complexed in the inner aqueous phase of the first emulsion. We observed a marked influence of microsphere composition on porosity, size distribution and PS-ODN encapsulation efficiency. Mainly, the presence of PEI induced the formation of large pores observed onto microsphere surface. Introduction of NaCl in the outer aqueous phase increased the encapsulation efficiency and reduced microsphere porosity. In vitro release kinetic of PS-ODN was also investigated. Clearly, the higher the porosity, the faster was the release and the higher was the burst effect. Using an analytical solution of Fick's second law of diffusion, it was shown that the early phase of PS-ODN and PS-ODN-PEI complex release was primarily controlled by pure diffusion, irrespectively of the type of microsphere. Finally, microspheres containing antisense TGF-β2 nanosized complexes were shown, after subconjunctival administration to rabbit, to significantly increase intracellular penetration of ODN in conjunctival cells and subsequently to improve bleb survival in a rabbit experimental model of filtering surgery. These results open up interesting prospective for the local controlled delivery of genetic material into the eye.

Pharmacologic and Pharmacokinetic Assessment of Anti-TGFβ2 Aptamers in Rabbit Plasma and Aqueous Humor

The aim of the study is to determine the bioactivity and effects of PEGylation on the pharmacokinetics in rabbit aqueous humor and plasma of an aptamer directed against TGFbeta2. Pharmacological activity of anti-TGFbeta2 aptamer in rabbit ocular fluid was demonstrated using a mink lung epithelial cell proliferation assay. For pharmacokinetic analyses, concentrations of aptamers in plasma and aqueous humor were determined over time following bilateral subconjunctival administration to Dutch-belted rabbits using a hybridization-based pseudo-enzyme-linked immunosorbent assay (ELISA) assay. Anti-TGFbeta2 aptamer (ARC81) binds to human TGFbeta2 with a K(D) of approximately 5 nM and inhibits the activity of human TGFbeta2 in vitro in a cell-based assay with an IC(50) of approximately 100 nM. ARC81 blocks endogenously derived TGFbeta2 in rabbit aqueous humor in vitro with an IC(50) of approximately 200 nM and an IC(90) of approximately 1 microM. In vivo in rabbit, ARC81 [no polyethylene glycol (PEG)] entered systemic circulation rapidly (t(max) = 1 h in plasma) relative to aptamer conjugates ARC117 (20 kDa PEG) and ARC119 (40 kDa PEG), which showed prolonged residence in the subconjunctival space and aqueous compartment (t(max) = 6 and 12 h, respectively, in plasma). Both 20- and 40-kDa aptamer conjugates reached maximal concentrations (C(max)) in aqueous humor of 23-30 nM and remained at or above 1 nM for as long as 12 h. Pharmacologically active levels of anti-TGFbeta2 aptamers can be sustained in the ocular fluid and local tissue environment over a 12-h period after single administration. Daily subconjunctival administration of PEGylated anti-TGFbeta2 aptamers should allow further pharmacological evaluation of these agents in a rabbit conjunctival scarring model. Perioperative administration, via subconjunctival injection, may prove to be an effective means to deliver therapeutic quantities of TGFbeta2 aptamer conjugates in trabeculectomy procedures.

Effect of pirenzepine on form deprivation myopia in chicks and its possible mechanism

To observe the effect of M1-selective muscarinic antagonist, pirenzepine, on form deprivation myopia and investigate the expression of MMP-2 and its inhibitor TIMP-2 in the fibrous sclera in order to better understand the mechanism by which pirenzepine inhibits myopia. 40 chicks after birth one day were divided into 4 groups randomly: I. Control group; II. Form deprivation group; III. Vehicle application group; IV. Pirenzepine injected group. Form deprivation myopia was established in right eyes of group II, III, IV by placement of a translucent occluder. The deprived eyes of group III and IV received daily subconjunctival administration of vehicle PBS and pirenzepine respectively. Optical measures such as refraction, axial length, equatorial diameter were made at the end of the experiment. Total RNA and protein were extracted from the posterior fibrous sclera chicks. The expression of MMP-2 and TIMP-2 mRNA and protein were investigated with RT-PCR and Western blot analysis respectively. Refraction status, axial length, equatorial diameter of the eyes in pirenzepine injected group were significantly lower when compared with form deprivation group (P < 0.01), but the parameters were higher when compared with normal control group therefore relatively myopic changes were detected. There were no significant difference between drug control and pirenzepine injected group when optical measures and the expression of MMP-2, TIMP-2 were concerned (P > 0.05). The expressions (mRNA and protein) of both MMP-2 and TIMP-2 were significantly different in form deprivation group when compared with normal control group (MMP-2 mRNA increased by 143.51%, P < 0.01; protein increased by 114.60%, P < 0.01; TIMP-2 mRNA decreased by 55.05%, P < 0.01; protein decreased by 53.73%, P < 0.01). In pirenzepine injected group the relative expression of MMP-2 mRNA and protein were decreased obviously by 41.95% (P < 0.01) and by 36.16% (P < 0.01), while TIMP-2 mRNA and protein expression was increased significantly by 72.46% (P < 0.01) and by 53.05% (P < 0.01) respectively compared with the form deprived group. Subconjunctivally administration of the M1 selective muscarinic antagonist, pirenzepine, partly prevents or restrains form deprivation induced myopia. It may exert its inhibitory effect by modulating the expression of MMP-2 and TIMP-2 in fibrous sclera.

Transscleral-RPE Permeability of PEDF and Ovalbumin Proteins: Implications for Subconjunctival Protein Delivery

To investigate the in vitro, ex vivo, and in vivo transscleral-retinal pigment epithelium (RPE) permeability of PEDF and structurally related proteins for the exploration of novel routes of protein delivery to the retina. Monkey RPE cells were cultured on permeable supports to separate apical and basal compartments. Porcine scleral tissue was placed in Ussing chambers to separate uveal and orbital compartments. Transepithelial resistance and voltage were measured by an electrical resistance system, and paracellular tracer flux was evaluated with trypan blue. Subconjunctival administration in rat eyes was by injections of soluble protein or by implantation of polyvinyl alcohol devices containing protein. Fluorescein-conjugated (Fl-) PEDF and ovalbumin were determined by spectrofluorometry, laser scanning, immunoblotting, epifluorescence, and confocal microscopy. Permeability was assessed by fluoresceinated-protein flux. Transepithelial resistance, impermeability to trypan blue, and confocal microscopy confirmed functional and structural tight junction formation of RPE cells cultured on permeable supports. Full-length Fl-PEDF and Fl-ovalbumin proteins diffused through RPE cell monolayers from either the apical or basal side. Fl-ovalbumin diffused through scleral tissues at constant rates. Subconjunctival Fl-PEDF or Fl-ovalbumin administration in vivo revealed movement of full-length protein into the choroid and retina as early as 1 hour. The sclera and RPE were permeable in vitro, ex vivo, and in vivo to PEDF and ovalbumin proteins. These large proteins can traverse through the sclera-RPE to reach the retina. In addition, these data prompt the proposal that subconjunctival protein delivery may represent a feasible and minimally invasive route for PEDF administration in the clinic.

Size-dependent disposition of nanoparticles and microparticles following subconjunctival administration

The purpose of this study was to determine the retention and ocular distribution of subconjunctivally administered nanoparticles and microparticles. Fluorescent polystyrene particles (carboxylate modified, negatively charged) of various sizes (20 nm, 200 nm and 2 microm; Fluospheres, dose 400 microg) were administered to male Sprague-Dawley rats by subconjunctival injection under anaesthesia. The disposition of the particles in the periocular and ocular tissues was studied for up to 60 days by quantifying the particle amounts using liquid extraction followed by spectrofluorimetric analysis. The effect of dose on the particle disposition was investigated with a 40-microg dose of the particles. The effect of an increase in surface hydrophobicity was evaluated for the 20 and 200 nm particles at 1 day post administration. Following periocular administration, penetration into the ocular tissues was negligible for the carboxylate-modified microparticles as well as nanoparticles. Almost the entire dose of the 200 nm and 2 microm particles was retained in the periocular tissue at 60 days post-administration. The 20 nm particles disappeared rapidly from the periocular tissue with 15 and 8% of administered dose remaining after 1 and 7 days, respectively. The 20 nm particles could not be detected in the periocular tissue at 60-days post-administration. An increase in the surface hydrophobicity did not affect the periocular retention of 200 nm particles but elevated that of the 20 nm particles, at the end of day 1. It was concluded that subconjunctivally administered 200 nm and larger particles can be almost completely retained at the site of administration for at least two months. Periocular administration of particulate systems of this size would likely be useful as sustained drug delivery systems.

Inhibitory effect of triamcinolone acetonide on corneal neovascularization

Corneal neovascularization (NV) plays an important role in the pathogenesis of corneal disorders. Recently, triamcinolone acetonide (TA) has been reported as a potential treatment for ocular angiogenesis. However, there are no reports on the inhibitory effect of TA on the corneal NV. Triamcinolone acetonide (2 mg) was administered to four rabbits' eyes by a subconjunctival injection immediately after a basic fibroblast growth factor (bFGF)-pellet was placed into the cornea. As a control, four eyes received an injection of distilled water. Four weeks later, the inhibition of corneal NV was evaluated as the percentage ratio of the vessel invasion area to the area that was sandwiched between the pellet and the limbus cornea. To identify the characteristic appearance of new corneal vessels, the control cornea was examined by using the antibody of vascular endothelial growth factor (VEGF). To confirm TA concentration in TA-treated corneas, the TA level was measured using high-performance liquid chromatography. Neovascularization from the limbus to the pellet was detected in control eyes 4 weeks after the bFGF pellet implantation. TA-treated eyes demonstrated the inhibition of the neovascular response to the pellet. The severity of NV as compared between control and TA-treated eyes was statistically significant (P<0.05). Morphologically, new vessel growth was shown in the control cornea, and endothelial cells of new vessels were positively stained with the antibody of VEGF. TA concentration in TA-treated corneas at 2 weeks showed 63.5+/-42.8 microg/g (n=4, mean +/- SD), while TA was not detected in control and TA-treated corneas at 4 weeks. The level of TA was effectively maintained for at least 2 weeks after the subconjunctival injection. We have demonstrated that subconjunctival TA administration inhibited rabbit corneal NV. This agent may prove useful in the treatment of corneal angiogenic disorders.

Subconjunctivally administered celecoxib-PLGA microparticles sustain retinal drug levels and alleviate diabetes-induced oxidative stress in a rat model

We have previously reported that repeated oral doses of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, reduced diabetes-induced retinal vascular endothelial growth factor (VEGF) expression [ Ayalasomayajula, S.P., Kompella, U.B., 2003. Celecoxib, a selective cyclooxygenase-2 inhibitor, inhibits retinal vascular endothelial growth factor expression and vascular leakage in a streptozotocin-induced diabetic rat model. Eur J Pharmacol 458, 283–289] and that retinal celecoxib delivery can be improved by several-fold following subconjunctival administration [ Ayalasomayajula, S.P., Kompella, U.B., 2004. Retinal delivery of celecoxib is several-fold higher following subconjunctival administration compared to systemic administration. Pharm Res 21, 1797–1804]. The objective of the current study was to determine whether polymeric microparticles of celecoxib sustain retinal drug levels following subconjunctival administration and alleviate diabetes-induced oxidative stress in a streptozotocin-induced diabetic rat model. Biodegradable poly (lactide-co-glycolide) (PLGA; 85:15) microparticles of celecoxib were prepared using solvent evaporation method and characterized for their size, morphology, encapsulation efficiencies, and in vitro release. The celecoxib-PLGA microparticles or solution containing 75 μg of celecoxib was administered subconjunctivally to one eye (ipsilateral) of Sprague Dawley rats and drug levels in the retina, vitreous, lens, and cornea of ipsilateral and contralateral eyes were determined on 1, 7, and 14 days using high-performance liquid chromatography (HPLC). The effect of subconjunctivally administered celecoxib-PLGA microparticles on oxidative stress in day 14 diabetic rat retinas was determined by measuring the retinal glutathione (reduced (GSH) and oxidized (GSSG)), thiobarbituric acid reactive substances, and 4-hydroxynonenal levels using spectrofluorometric and colorimetric methods. Solvent evaporation method produced spherical celecoxib-PLGA microparticles with mean diameters of 3.9±0.6 μm and 68.5% loading efficiency. These microparticles sustained celecoxib release during the 49-day in vitro release study. Subconjunctivally administered celecoxib-PLGA microparticles sustained retinal and other ocular tissue drug levels during the 14-day study in rats. No detectable celecoxib levels were observed in the contralateral eye. The celecoxib-PLGA microparticles significantly inhibited the diabetes-induced increases in thiobarbituric acid reactive substance ( P=0.012) and 4-hydroxynonenal levels ( P=0.029). The particles also inhibited the GSH depletion and the increase in GSSH/GSH ratio associated with diabetes but the effects were not statistically significant ( P=0.12). Thus, following subconjunctival administration, celecoxib-PLGA microparticles sustained retinal celecoxib delivery and inhibited diabetes-induced retinal oxidative damage, indicating their potential usefulness in treating diabetes-induced retinal abnormalities.

Precipitate Formation after Subconjunctival Administration of Ceftazidime and Vancomycin

This is a case report of precipitate formation after the administration of subconjunctival antibiotics after uneventful intravitreal antibiotic administration in a patient with endophthalmitis.

Retinal Delivery of Celecoxib Is Several-Fold Higher Following Subconjunctival Administration Compared to Systemic Administration

We have previously demonstrated that celecoxib, a selective COX-2 inhibitor, reaches the retina following repeated oral administrations and inhibits diabetes-induced vascular endothelial growth factor (VEGF) mRNA expression and vascular leakage in a rat model. The aim of this study was to quantify the relative retinal bioavailability of celecoxib from the subconjunctival route compared to a systemic route. The plasma and ocular tissue distribution of celecoxib was determined in male Sprague-Dawley rats following subconjunctival and intraperitoneal administrations of drug suspension at a dose of 3 mg/rat. The animals were sacrificed at 0.5, 1, 2, 3, 4, 8, and 12 h post-dosing, the blood was collected, and the eyes were enucleated and frozen. The plasma, sclera, retina, vitreous, lens, and the cornea were isolated and celecoxib levels were determined using an HPLC method. The tissue exposure of the drug was measured as the area under the curve (AUC(0-infinity)) of the concentration vs. time profiles. The relative bioavailability was estimated as the AUC(0-infinity) ratio between subconjunctival and intraperitoneal groups. For the subconjunctivally dosed (ipsilateral) eye, the AUC(0-infinity) ratios between subconjunctival and intraperitoneal groups were 0.8 +/- 0.1, 53 +/- 4, 54 +/- 8, 145 +/- 21, 61 +/- 16, and 52 +/- 6 for plasma, sclera, retina, vitreous, lens, and cornea, respectively. For the contralateral ocular tissues, the AUC0-infinity ratios were 1.2 +/- 03, 11 +/- 0.3, 1.1 +/- 0.4, 1.0 +/- 0.3, and 1.2 +/- 0.3 in the sclera, retina, vitreous, lens, and the cornea, respectively, between the subconjunctival and the intraperitoneal groups. Assuming that the drug AUCs in contralateral eye were equal to the systemic pathway contribution to AUCs in the ipsilateral eye, the percent contribution of local pathways as opposed to systemic circulation for celecoxib delivery to the ipsilateral eye tissues was estimated to be 98% or greater. The retinal delivery of celecoxib was substantially higher following subconjunctival administration compared to the intraperitoneal route. The transscleral pathway almost completely accounts for the retinal celecoxib delivery following subconjunctival administration.

Subconjunctival nano- and microparticles sustain retinal delivery of budesonide, a corticosteroid capable of inhibiting VEGF expression.

The purpose of this study was to determine whether budesonide inhibits expression of vascular endothelial growth factor (VEGF) in a retinal pigment epithelial cell line (ARPE-19) and to determine whether subconjunctivally administered budesonide nano- and microparticles sustain retinal drug levels. The effect of budesonide (100 pM to 10 microM) on VEGF secretion, expression of VEGF mRNA, and cytotoxicity were determined in ARPE-19 cells by ELISA, RT-PCR, and a cell-viability assay, respectively. To determine the involvement of glucocorticoid receptor in the observed effects of budesonide, secretion and mRNA expression studies were also performed in the presence of a glucocorticoid receptor antagonist (RU486). DL-Polylactide (PLA) nano- and microparticles containing budesonide were prepared by a solvent evaporation technique, and the particles were characterized for size, morphology, encapsulation efficiency, and in vitro release. Budesonide-PLA nano- and microparticles were administered subconjunctivally to one eye of Sprague-Dawley rats and drug levels in the retina, vitreous, lens, and cornea of both eyes were determined at the end of 1, 7, and 14 days. At concentrations devoid of cytotoxicity, budesonide inhibited VEGF secretion as well as mRNA expression in ARPE-19 cells in a dose-dependent manner. RU486 treatment prevented budesonide-mediated inhibition of VEGF secretion and VEGF mRNA expression. Budesonide-PLA nano- (345 nm) and microparticles (3.6 microm), with an encapsulation efficiency of 65% and 99%, respectively, sustained budesonide release in vitro. After subconjunctival administration, both budesonide-PLA nano- and microparticles produced sustained budesonide levels in the retina and other ocular tissues. Budesonide is capable of inhibiting VEGF expression through glucocorticoid receptor activity. Subconjunctivally administered budesonide-PLA nano- and microparticles sustain retinal drug delivery.

Effects of orally administered olopatadine hydrochloride on the ocular allergic reaction in rats

Olopatadine hydrochloride is an antiallergic agent with histamine H 1 receptor antagonistic action. We investigated the effects of olopatadine on passive anaphylaxis reaction- and compound 48/80-induced conjunctivitis in rats. Allergic conjunctivitis was induced in rats passively sensitized by injection of rat anti-ovalbumin (anti-OVA) serum into the upper subconjunctiva of the right eye, followed by intravenous administration of the antigen and Evans blue dye. After 30 min, the amount of dye leaking into the conjunctiva was measured. Non-allergic conjunctivitis was induced in rats by injection of compound 48/80. Olopatadine or other reference compounds were orally given 1 h before the challenge. The amount of dye leaking into the conjunctiva following passive anaphylaxis was significantly inhibited by oral administration of 0.01-1 mg/kg olopatadine and the ID 50 value was 0.093 mg/kg. In the control group, pathological examination revealed edema and lymphocyte infiltration in the conjunctiva and the palpebral skin. Olopatadine, at 0.03 and 0.3 mg/kg, reduced the grade of these pathological findings. The other antiallergic drugs (1 mg/kg loratadine, 0.3 mg/kg epinastine, 0.3 mg/kg cetiridine, 1 mg/kg ebastine, 30 mg/kg fexofenadine and 3 mg/kg chlorpheniramine), when administered orally, inhibited the passive anaphylaxis reaction-induced vascular hyperpermeability of the conjunctiva, as was the case with olopatadine hydrochloride. Subconjunctival administration of compound 48/80 induced vascular hyperpermeability, which is presumably mediated by histamine release. Oral administration of 0.1 and 1 mg/kg olopatadine significantly inhibited the amount of dye leakage. Orally administered olopatadine is expected to improve allergic conjunctivitis.