Intraocular properties of a repository urokinase receptor antagonist a36 Peptide in rabbits.

PURPOSE: To evaluate the intraocular properties of A36, a peptide that directly antagonizes the cell surface urokinase receptor and so prevents pericellular urokinase plasminogen activator activity. METHODS: A total of 41 rabbits were used. The toxicity study tested three doses of A36: 1 mg/ eye, 0.3 mg/eye, and 0.1 mg/eye. At 2 and 12 weeks, eyes were evaluated by ERG and histology. Pharmacokinetics were studied in rabbit eyes with the dose of 1 mg/eye in two different formulations: a micronized preparation and a non-micronized formulation. Eyes were enucleated at months 1, 2, 3, 4, and 5. Vitreous, retina, and choroid were collected separately for active A36 analysis. RESULTS: We did not find ocular toxicity with low and medium doses. At the highest dose, there was a transient toxicity at 2 weeks but was not notable at 3 months. The target choroid concentration of A36 was chosen as > or =100 nM. The micronized formulation at months 1, 2, and 3 combined, showed variable levels in the choroid giving 5/10 (50%) of the therapeutic level; the non-micronized formulation at months 4 and 5 combined, gave 6/7 (86%) of the therapeutic level, although this difference was not statistically significant. CONCLUSION: A36 appears to be long lasting; the non-micronized formulation of A36 gave concentrations above therapeutic level in the choroid at months 4 and 5. Optimization of the formulation of A36, particularly the particle size, may result in a promising new compound for exudative age-related macular degeneration treatment.

Characterization of cytokine gene expression associated with noninfectious human immunodeficiency virus retinopathy in human autopsy eyes.

PURPOSE: The purpose of this study was to determine the cytokine-related pathogenesis of human immunodeficiency virus retinopathy in human autopsy eyes. METHODS: Fresh autopsy eyes were procured from clinically diagnosed patients with acquired immunodeficiency syndrome who had died as a result of disease-related complications; eyes were immediately immersed in RNAlater. Clean 2-mm trephines were used to punch individual pathologic retina in areas of cotton-wool spots and control punches. Total RNA was extracted using the TRIzol extraction protocol, and the optimal density of the RNA was measured at an optical density of 260 nm. [Delta]Ct (cytokine) values were calculated using the comparative cytokine analysis method. The results are expressed as a mean fold modulation and as a statistical comparison of Ct values controlling for retinal areas without a lesion in the same eye. RESULTS: The fold modulations and the statistical comparisons of the cytokines studied in tissues from cotton-wool spots and control retina, respectively, regulated on activation normal T cell expressed and secreted (RANTES), macrophage inflammatory protein 1beta, macrophage inflammatory protein 1alpha (5.32x, P = 0.04), and Bcl-2-associated X protein (1.24x, P = 0.05) had a marked elevation of fold modulation and were statistically significant compared with control tissue. Interleukin-8 (1.09x, P = 0.18), interleukin-4, and interleukin-10 (2.7x, P = 0.30) were not significantly expressed in cotton-wool spots. CONCLUSION: Certain inflammatory human immunodeficiency virus-associated and apoptotic cytokines are expressed in cotton-wool spots in eyes with human immunodeficiency virus retinopathy.

Quantum dot labeling and imaging of glial fibrillary acidic protein intermediate filaments and gliosis in the rat neural retina and dissociated astrocytes.

The use of antibody and peptide functionalized semiconductor quantum dots holds considerable potential for specific labeling of target antigens and high resolution optical imaging of biological preparations. Despite this potential, their use in neuroscience is not yet widespread; a number of technical and methodological challenges must still be overcome in order to produce reliable and reproducible labeling protocols. We have optimized and used anti-GFAP functionalized quantum dots for specific labeling of intermediate filaments in astrocyte and Müller glial cells in sections of intact rat neural sensory retina and dissociated primary spinal cord astrocytes. These techniques produced stable and robust imaging of retinal astrocytes and Müller cells with minimal non-specific background labeling and intense fluorescence resulting in a high signal to noise ratio. This resulted in clear and efficient labeling of normal levels of GFAP in the retina and the ability to differentiate it from pathologically high levels of GFAP associated with reactive gliosis in a laser induced injury model. Labeling and imaging of dissociated astrocytes demonstrated the presence of what appeared to be highly complex organizations of fine intermediate filaments that spanned between cells to form intricate networks of filamentous intercellular bridges. The presence of these structures in situ and in vivo as well as any potential functions remain to be determined, but their identification should be greatly facilitated by quantum dot labeling protocols.

Toxicity and intraocular properties of a novel long-acting anti-proliferative and anti-angiogenic compound IMS2186.

PURPOSE: To investigate the intraocular properties and toxicity of IMS2186, a small molecule developed as an anti-choroidal neovascularization (anti-CNV) drug. MATERIALS AND METHODS: Cellular toxicity and mechanism of action was tested on cell lines in vitro. Intraocular studies used rabbits for drug dissolution as well as toxicity and rats for the treatment study as well as the toxicity confirmation study. Rabbits' eyes were injected with 2.5 mg of IMS2186 and observed for 36 weeks. Laser-induced CNV in rats was treated with IMS2186, Kenalog, or phosphate-buffered saline (pBS). Fluorescein angiography (FA) and immunohistochemical processing of the globes was performed. RESULTS: The anti-proliferative IC(50) of IMS2186 for human fibroblast cells was 1.0-3.0 microM and 0.3-3.0 microM for human cancer cells; the IC(50) of IMS2186 to inhibit endothelial tube formation was 0.1-0.3 microM. The IC(50) of IMS2186 for inhibiting the production of pro-inflammatory cytokines was 0.3-1 microM. The IC(50) of IMS2186 for inhibiting macrophage migration was 1 micrM. These biological properties were not species specific. IMS2186 can be formulated as a suspension for long-lasting release and when delivered intraocularly, no intraocular toxicity was observed by slit lamp exam, fundus exam, intraocular pressure measurements, or by electroretinography. FA showed a reduction in the leakage in eyes treated with IMS2186 and triamcinolone acetonide; DAPI staining also showed significantly less cellularity in IMS2186-treated lesions as compared to PBS (p = 0.0025). CONCLUSION: IMS2186 may be a safe intraocular therapeutic agent for intraocular proliferation and angiogenesis.

Intraocular properties of an alkoxyalkyl derivative of cyclic 9-(S)-(3-hydroxyl-2-phosphonomehoxypropyl) adenine, an intravitreally injectable anti-HCMV drug in rabbit and guinea pig.

PURPOSE: The aim of this study was to investigate intraocular properties and determine the highest nontoxic dose of hexadecyloxypropyl-cyclic-HPMPA (HDP-cHPMPA), a novel, potent, intravitreally injectable, slow-releasing crystalline drug for long-acting treatment of cytomegalovirus (CMV) retinitis. METHODS: Various concentrations of HDP-cHPMPA were first studied in vitro in a human foreskin fibroblast (HFF) cell line infected with human cytomegalovirus (HCMV) to determine the EC50. In vivo, 9 pigmented rabbits and 3 doses (55, 100, and 550 microg/eye) were tested in triplicate in 1 eye of each animal. The eyes were monitored with slit lamp, tonopen, indirect ophthalmoscopy, electroretinography (ERG), and histology. A confirmation toxicity study with the dose equivalent to the highest nontoxic dose in rabbit was performed in 9 guinea pig eyes (a second species) to study the potential adverse effect on intraocular pressure (IOP). RESULTS: In vitro testing in HFF cells showed an EC50 against HCMV of 0.02 microM, which is 75- and 60-fold greater than that of ganciclovir and cidofovir, respectively. All eyes injected with 550 microg/eye and 1 eye injected with 100 microg/eye of HDP-cHPMPA showed toxicity clinically (e.g., vitreous cells, disc edema, and retinal inflammation) as well as histologically (e.g., inflammatory cells in iris, vitreous, and retinal layers with disorganization). None of the eyes injected with 55 microg/eye of HDP-cHPMPA showed toxicity clinically (including ERG) and histologically. The equivalent dose (9.2 microg/eye) in the guinea pig eyes did not show toxicity either, including IOP evaluation (P > 0.05 at all time points after injection). CONCLUSIONS: Intravitreal injection of the highest nontoxic dose of 55 microg/eye of HDP-cHPMPA in rabbit eyes yields a calculated intravitreal concentration of 65 microM, which is 3250-fold greater than the EC50 against HCMV (0.02 microM). Also, it does not cause hypotony in rabbit and guinea pig eyes and has a vitreous residence time of over 4 months.