Efficacy of Antibody Delivery to the Retina and Optic Nerve by Topical Administration.

PURPOSE: The purpose of this study was to determine whether nonspecific and ICAM-1-specific IgG1 antibodies can accumulate in the rat retina following topical application, and to develop a model system to show that antibodies that reach the posterior segment retain their pharmacological properties. METHODS: Eye drops containing mouse IgG1 or anti-ICAM-1 and the permeation enhancer saponin were topically applied to the eyes of Lewis rats. Concentrations were determined in the retina and optic nerve up to 30 min later using ELISA assays. We also developed an in vitro model to assess the pharmacologic activity of topically delivered antibodies in the retina based on the requirement of human umbilical vein endothelial cells (HUVECs) for vascular endothelial growth factor (VEGF) for growth. Rat eyes were treated with anti-VEGF antibody in the same manner as above; their retinas, harvested shortly thereafter, were added to HUVECs cultured in VEGF-containing media. The effect of these retinal homogenates on HUVEC proliferation was then assessed. RESULTS: Significant concentrations of IgG1 were detected in the optic nerve (P < 0.001) and retina (P < 0.0001) following topical application. Anti-ICAM-1 antibody also accumulated in the retina after topical application, though levels were less than those seen with IgG1 probably owing to a lower starting concentration. Retinal homogenates from eyes treated with anti-VEGF antibody significantly suppressed HUVEC proliferation (P < 0.0001). CONCLUSIONS: Our data support the contention that topically applied antibodies can accumulate in the posterior segment, and suggest they retain their pharmacological properties.

Selective toxicity of rose bengal to ovarian cancer cells in vitro.

Rose bengal (RB) has been utilized as a photodynamic agent for the targeted killing of cancer cells. Recent data suggest that intralesional RB alone may be effective in chemoablating locoregional and metastatic melanomas. The ability of RB to induce direct and bystander melanoma cell death led to the speculation that it may be similarly effective in the treatment of other neoplasms. The objective of this study was to determine whether RB can limit the growth, or kill, ovarian cancer cells in vitro. Ovarian carcinoma cells with or without a germline BRCA1 mutation were cultured with up to 800 µM RB for one hour or four days, after which their ability to proliferate was assessed using the MTT assay. Control cells included an embryonic kidney cell line transformed with adenovirus, and normal human fibroblasts. Ovarian cancer cells exhibited significant dose-dependent suppression of growth in response to RB; this suppression was similar to that seen with carboplatin. RB treated ovarian cancer cells appeared rounded, shrunken, and damaged. RB also inhibited the growth of kidney tumor cells but was much less effective in slowing the growth of normal human fibroblasts suggesting that RB-mediated growth suppression might be tumor cell specific. Ovarian cancer cells treated with RB displayed a significant increase in apoptosis that peaked at approximately four times the levels seen in untreated control cells. Furthermore, RB exposure resulted in the intracellular generation of reactive oxygen species (ROS) at levels that were significantly greater than in untreated cells and similar to levels seen in cells treated short term with H(2)O(2). These data suggest that RB may not only suppress ovarian cancer cell growth but also induce their apoptotic cell death, justifying the further investigation of the effects of RB in an animal model of ovarian cancer.

Long-term, topical insulin administration increases the severity of retinal vascular pathology in streptozotocin-induced diabetic rats.

BACKGROUND: We previously reported that insulin accumulated in the retina of diabetic rats after topical insulin eye drop treatment. In light of insulin's reported benefits to the retina, we hypothesized that the delivery of insulin to the retina by eye drop application might be efficacious in preventing diabetic retinopathy. METHODS: We applied daily, uni-ocular porcine insulin drops (0.75%) to diabetic rats for 14 months and then analyzed their retinas for vascular pathology. RESULTS: Our data showed that high-dose insulin eye drop treatment increased the number of retinal acellular capillaries, with many of these capillaries exhibiting a degenerated, threadlike appearance. The retinas also showed extensive capillary obliteration and had tangled masses of vascular cells. The glycated hemoglobin levels of eye drop-treated rats were similar to those found in control, vehicle-treated diabetic animals at sacrifice. Retinal insulin levels remained elevated after a 2-week regimen of daily insulin eye drops, suggesting that our treatment protocol resulted in the pooling of insulin in the retina. A similar treatment regimen was also found to have no effect on retinal glucose concentration. CONCLUSION: Our results showed that treatment of diabetic rats with daily, high-dose insulin eye drops intensified their retinal pathology.

Ocular involvement in patients infected by the West Nile virus.

West Nile virus (WNV), a mosquito-borne RNA virus for which there is no treatment, began emerging as a threat to health in the United States in 1999. Since then, its frequency and apparent clinical severity have increased. Patients with severe disease may experience ocular complications that include pain, vitreous inflammation, nonrelapsing chorioretinitis, retinal vasculitis, chorioretinal scarring, optic neuritis, and retinal hemorrhages. Age appears to be a risk factor for the development of ocular symptoms. Most patients with ocular involvement report floaters or decreased vision. Many ocular inflammatory conditions with an associated underlying systemic cause often present with chorioretinitis. However, the characteristic distribution and linear array or scattered pattern of the chorioretinal lesions seen in WNV is distinctive. The foregoing notwithstanding, other multifocal choroidopathies must be considered including syphilis, ocular histoplasmosis, multifocal choroiditis, tuberculosis, and sarcoid. Although topical steroids were reported to resolve WNV-induced uveitis and its associated keratic precipitates, most cases resolved irrespective of treatment, and relapses were uncommon. It is important for the eye care professional to be alert to the possible presence of WNV, particularly in older patients who present with ocular symptoms during mosquito season. Thus, a thorough ocular evaluation should include a dilated fundus examination and, when indicated, fluorescein angiography should be performed in patients suspected of being infected with WNV.

Pharmacokinetics of insulin uptake by ocular tissues and the role of cerebrospinal fluid in optic nerve insulin accumulation following topical insulin application.

BACKGROUND: As part of our ongoing studies concerning the efficacy of using topically applied medications to treat retinal and optic nerve diseases, we previously showed that insulin accumulated in the retina, optic nerve, and cerebrospinal fluid (CSF) following topical application. The purpose of this study was to investigate which route insulin takes to get to the posterior segment of the eye, and to specifically assess the role of the CSF in optic nerve insulin accumulation. METHODS: Lewis rats that received 125I-insulin eye drops were killed at different time points and their ocular tissues counted in a gamma counter. In order to determine whether elevated levels of CSF insulin could lead to optic nerve insulin accumulation, a separate cohort of animals was injected in their lumbar cistern with unlabeled insulin and their optic nerves later assessed for the presence of insulin by enzyme-linked immunosorbent assay. RESULTS: All ocular tissues (except the lens) showed at least one significant time point elevation in 125I-insulin compared to baseline. Both the combined cornea/iris/ciliary body and the sclera showed a notable, overall increase in counts over baseline, with values at 20 and 30 minutes being significantly elevated. The highest numbers of counts were seen in the aqueous humor. Animals injected intralumbar cisternally with insulin showed elevated insulin concentrations in their optic nerves and cisterna magna-derived CSF that did not appear to be due to uptake of insulin from the circulation. CONCLUSIONS: These results support the hypothesis that the insulin that accumulates in the retina and optic nerve following topical application arrives there after diffusing through the sclera, though an intraocular route--while unlikely--cannot be ruled out.

Accumulation of porcine insulin in the rat brain and cerebrospinal fluid following ocular application.

We previously reported that insulin accumulated in the retina and optic nerve following ocular application. Since the optic nerve is surrounded by meninges and cerebrospinal fluid (CSF) and since it extends back to the thalamus, we examined whether the topical application of insulin eye drops also resulted in the accumulation of insulin in the CSF and brain. The data presented in this paper show that this is in fact the case. Following the ocular application of a 0.75% solution of porcine insulin, significant concentrations of insulin were demonstrable in the CSF extracted from the cisterna magnum, as well as in three brain regions. While it is not yet clear how insulin got into these target tissues, our data argue against a mechanism involving uptake from the blood (a fraction of topically applied compounds normally enters the vasculature through the conjunctiva and nasal mucosa). It is theorized that insulin may enter the CSF surrounding the optic nerve and by so doing, not only disseminate throughout the CSF space but also throughout the brain. The implications of these findings for central nervous system drug delivery are discussed.

Pharmacokinetics of topical ocular drug delivery: potential uses for the treatment of diseases of the posterior segment and beyond.

In developing a drug delivery strategy, issues of absorption, distribution, metabolism, and elimination must be considered. The eye presents unique opportunities and challenges when it comes to the delivery of pharmaceuticals, and is most accessible to the application of topical medications. While absorption by this route is inefficient, there are few side effects. While it has been assumed that topically applied drugs penetrated into the intraocular environment through the cornea, this is currently being reassessed. More recent investigations have shown that the conjunctival route of entry plays an important role in the penetration of drugs into the anterior segment. Furthermore, topically applied drugs have been shown to have access to the sclera from the conjunctiva. As such, it is conceivable that such drugs could find their way to the posterior segment. Data suggest that the sclera is readily permeable to even large molecular weight compounds ( approximately 150 kD). The recent finding that topically applied nepafenac inhibited choroidal and retinal neovascularization by decreasing the production of VEGF, as well as our data showing that even a large molecular weight peptide like insulin can accumulate in the retina and optic nerve after topical application, supports the contention that topically applied drugs can not only reach the posterior segment, but that they can also be therapeutic. Finally, the implications of our findings that topically applied insulin also accumulates in the contralateral eye as well as in the central nervous system are discussed.

Insulin and tropicamide accumulate in the contralateral, untreated eye of rats following ipsilateral topical administration by a mechanism that does not involve systemic uptake.

BACKGROUND: This study was designed to determine: (1) whether the accumulation of insulin in the contralateral retina and aqueous humor following ipsilateral topical insulin administration was due to systemic uptake and (2) whether tropicamide, applied to one eye, could induce dilation in the contralateral eye by a mechanism that did not involve systemic uptake. METHODS: Insulin eye drops were applied to the left eye of intact and decapitated rats, and their retinas and aqueous humors were then removed and their insulin levels quantified. In a separate experiment live animals received 0.1% tropicamide in their left eye and had their pupillary dilation response in both eyes measured at different time points. RESULTS: Administration of insulin to the left eye of decapitated rats resulted in its significant accumulation not only in the left retina and aqueous humor, but also in the retina and aqueous humor of the right eye. Similar aqueous humor results were obtained when live animals were used. Tropicamide drops induced marked pupillary dilation in treated eyes; the pupils of the contralateral, untreated eyes also dilated significantly, but less than did the treated pupils. The pupils of rats injected with tropicamide intravenously showed negligible dilation. CONCLUSIONS: These results showed that insulin accumulated in the retina and aqueous humor of contralateral, untreated eyes following topical application, by a mechanism that did not appear to involve systemic uptake. Similarly, tropicamide provoked a dilation response in the unheated eye by a mechanism that similarly did not appear to involve uptake from the blood.

Penetración ocular de fármacos por vía no corneal. Nuevas tendencias / Trends in the noncorneal delivery of drugs into the eye

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Accumulation of topically applied porcine insulin in the retina and optic nerve in normal and diabetic rats.

PURPOSE: To explore the pharmacokinetics of topical insulin administration in relation to retinal and optic nerve retention. METHODS: Insulin eye drops (approximately 15 microL: 0.75% porcine insulin + 0.5% permeation enhancer) were applied to the eyes of normal and diabetic rats. The rats were killed at various intervals up to 16 hours, and the retinas and optic nerves from both eyes were analyzed for the presence of insulin in an ELISA. The extent to which systemically absorbed insulin accounted for the findings of insulin in the retina was explored by examining the effects of intravenously injected insulin on retinal insulin levels and by examining the effects of eye drop administration in decapitated rats. RESULTS: Insulin levels rose significantly and peaked in the retina of normal rats 20 minutes after eye drop application (0.7 pg/microg; P < 0.00001). Levels in diabetic retinas peaked at 60 minutes (0.66 pg/microg; P < 0.004) and remained elevated for a longer period than in normal rats. The contralateral retina showed delayed accumulation of lesser amounts of insulin in both normal and diabetic rats. Significant elevations also occurred in the optic nerves in normal and diabetic rats, with concentrations reaching 13 pg/microg in normal rats at 20 minutes and 26 pg/microg in diabetic rats at 5 hours. Topical insulin application resulted in a decrease in serum glucose concomitant with an increase in serum porcine insulin. It did not appear, however, that the systemic absorption of insulin contributed to the accumulation of insulin in the ipsilateral retinas, for two reasons: The intravenous injection of a high concentration of insulin did not appreciably influence retinal insulin levels, and the application of insulin eye drops to decapitated rats still resulted in the accumulation of insulin in the retina. CONCLUSIONS: These results led to the conclusion that topically applied insulin accumulates in the retina and optic nerve in normal and diabetic rats, with levels remaining elevated longer in diabetic animals. It did not appear that systemically absorbed insulin, resulting from ocular drainage, contributed to this effect.