Long-acting protein drugs for the treatment of ocular diseases.

Protein drugs that neutralize vascular endothelial growth factor (VEGF), such as aflibercept or ranibizumab, rescue vision in patients with retinal vascular diseases. Nonetheless, optimal visual outcomes require intraocular injections as frequently as every month. Here we report a method to extend the intravitreal half-life of protein drugs as an alternative to either encapsulation or chemical modifications with polymers. We combine a 97-amino-acid peptide of human origin that binds hyaluronan, a major macromolecular component of the eye's vitreous, with therapeutic antibodies and proteins. When administered to rabbit and monkey eyes, the half-life of the modified proteins is increased ∼3-4-fold relative to unmodified proteins. We further show that prototype long-acting anti-VEGF drugs (LAVAs) that include this peptide attenuate VEGF-induced retinal changes in animal models of neovascular retinal disease ∼3-4-fold longer than unmodified drugs. This approach has the potential to reduce the dosing frequency associated with retinal disease treatments.

AAV-mediated RLBP1 gene therapy improves the rate of dark adaptation in Rlbp1 knockout mice.

Recessive mutations in RLBP1 cause a form of retinitis pigmentosa in which the retina, before its degeneration leads to blindness, abnormally slowly recovers sensitivity after exposure to light. To develop a potential gene therapy for this condition, we tested multiple recombinant adeno-associated vectors (rAAVs) composed of different promoters, capsid serotypes, and genome conformations. We generated rAAVs in which sequences from the promoters of the human RLBP1, RPE65, or BEST1 genes drove the expression of a reporter gene (green fluorescent protein). A promoter derived from the RLBP1 gene mediated expression in the retinal pigment epithelium and Müller cells (the intended target cell types) at qualitatively higher levels than in other retinal cell types in wild-type mice and monkeys. With this promoter upstream of the coding sequence of the human RLBP1 gene, we compared the potencies of vectors with an AAV2 versus an AAV8 capsid in transducing mouse retinas, and we compared vectors with a self-complementary versus a single-stranded genome. The optimal vector (scAAV8-pRLBP1-hRLBP1) had serotype 8 capsid and a self-complementary genome. Subretinal injection of scAAV8-pRLBP1-hRLBP1 in Rlbp1 nullizygous mice improved the rate of dark adaptation based on scotopic (rod-plus-cone) and photopic (cone) electroretinograms (ERGs). The effect was still present after 1 year.

Complement deposition and microglial activation in the outer retina in light-induced retinopathy: inhibition by a 5-HT1A agonist.

PURPOSE: Increasing evidence supports a role for complement in the pathogenesis of age-related macular degeneration (AMD). This study evaluated retinal microglia, T-lymphocytes, and complement deposition in a light-induced retinopathy model. The effect of a serotonin (5-hydroxytryptamine, 5-HT(1A)) agonist on these processes was investigated. METHODS: Rats were dark adapted for 24 hours before a 6-hour blue light exposure. Some animals were predosed subcutaneously with AL-8309A. Retinas were evaluated at different times after light exposure. Paraffin sections were stained with antibody for a microglial marker (Iba1), a T-lymphocyte marker (CD3), and complement components C1q, C3, factor B, factor H, and membrane attack complex (MAC). RESULTS: Light exposure resulted in substantial photoreceptor and RPE loss. Robust microglia activation and migration to the outer retina occurred rapidly. Substantial T-lymphocyte recruitment did not occur. Complement alternative pathway was strongly activated, resulting in the deposition of C3, factor B, factor H, and MAC in the area of photic lesions. Dosing with AL-8309A prevented retinal lesions and decreased microglia activation/recruitment and complement deposition in the outer retina. CONCLUSIONS: In blue light exposed retinas, microglia were activated and migrated toward the outer retina, whereas a T-lymphocyte response was minimal. The innate immune system was markedly activated, with substantial complement deposition in the outer retina after light exposure. This complement deposition was prevented by AL-8309A. This model may be useful in the evaluation of complement inhibitors and other neuroprotectants intended for ocular use. AL-8309 is under evaluation in the clinic and may be useful in the treatment of AMD.

Immunofluorescent labeling of proteins in cultured cells with quantum dot secondary antibody conjugates.

Our understanding of the level and distribution of gene and protein expression in cells is a key component of modern cell biological and medical research. Detecting intracellular proteins with labeled antibodies or genes with labeled oligonucleotide sequences by fluorescence microscopy requires fixation of the target molecule in its natural distribution and the penetration of the probe to the target. This typically involves chemical fixation followed by a detergent treatment that renders the cell membrane permeable to a labeling antibody. The advantages of using quantum dots (QDs) over organic dyes to detect expression, such as high brightness, stability, and simplified multiple target labeling has been described in previous publications. However, QDs are structurally larger than organic dye probes and require different fixation and permeabilization conditions for optimum labeling. In the chapter, we describe several protocols for labeling proteins in nuclear, cytoplasmic, and membranous compartments with QD conjugates.

Quantitative ex vivo detection of rodent retinal ganglion cells by immunolabeling Brn-3b.

To evaluate the neuroprotective potential of drug candidates to treat human glaucoma, a short-term rodent model of retinal ganglion cell death was employed. Transient ischemia applied to the rodent retina, with subsequent reperfusion for 1-4 weeks, produces an experimental retinal ganglion cell death that is quantifiable. A widely used method to detect viable retinal ganglion cells involves surgical injection of labeling compounds into the superior colliculus of the rodent brain, the retrograde transport of the compounds along the axons to the retina, and subsequent microscopic evaluation of the retina. In order to circumvent the labor intensive and invasive surgery of this method, we sought an alternative means of assessing retinal ganglion cell survival that would be more suitable for high-throughput analysis. We therefore developed a method of immunolabeling whole retinas ex vivo with an antibody to Brn-3b, an antigen expressed in a subpopulation of retinal ganglion cells, that allows for detection of a representative retinal ganglion cell population. Fluorescently tagged Brn-3b immunolabeled retinas were flat-mounted, digitally imaged, and assessed using image analysis software. We determined that 60 min of ischemia caused a 49% and a 32% decrease in Brn-3b positive retinal ganglion cells in Lewis rats after 4 weeks reperfusion, and Sprague-Dawley rats after 2 weeks reperfusion, respectively. In Swiss Webster ND4 mouse retinas subjected to 45 min ischemia and 7 days reperfusion, we found a 70% decrease in Brn-3b positive cells. Thus, ex vivo immunolabeling of retinal ganglion cells using antibody to Brn-3b provides an alternative to other methods of quantifying retinal ganglion cells.

Selective cyclooxygenase-2 inhibition does not alter keratinocyte wound responses in the mouse epidermis after abrasion.

The cyclooxygenase isoforms, COX-1 and COX-2, are the rate limiting enzymes in the biosynthesis of prostaglandin E(2), a major prostaglandin involved in epidermal homeostasis and repair. Epidermal injury results in transient hyperplasia and induction of COX-2 expression. The role of COX-2 in this hyperplasia is unknown, however. In this study, we characterized the epidermal expression of COX isozymes following wounding by abrasion in SKH-1 mice using immunohistochemistry, in situ hybridization, and Western analysis. In addition, we evaluated pivotal keratinocyte functions necessary for the reparative hyperplasia, including proliferation by 5-bromo-2'deoxy-uridine labeling and differentiation by the expression of involucrin, keratin 1, and keratin 6. Although COX-1 expression in keratinocytes remained unchanged during wound healing, COX-2 expression was induced coincidentally with keratinocyte proliferation and keratin 6 expression, suggesting a role for COX-2 in epidermal repair. The role of COX-2 was also evaluated using the selective COX-2 inhibitor SC-791 and the traditional COX inhibitors indomethacin and diclofenac. Neither inhibitor altered keratinocyte proliferation or differentiation following abrasion, in contrast to dexamethasone, which delayed these responses. Our results indicated that, although COX-2 expression was coincident with transient epidermal hyperplasia and keratinocyte proliferation/differentiation during the healing of epidermal injury, it does not play a pivotal role in this repair process.

Direct evidence for a role of cyclooxygenase 2-derived prostaglandin E2 in human head and neck xenograft tumors.

Both nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase (COX) 2-selective inhibitors such as celecoxib are being reported as having potent anticancer activity in laboratory models. Several reports have suggested that the mechanism of action of these agents in reducing tumor volume/burden is unrelated to their inhibition of prostaglandin synthesis. Many in vitro reports use supraphysiological concentrations of these drugs to demonstrate COX-independent activities on apoptosis or proliferation. In vivo, most murine tumor models express COX-2 only in the vasculature and stroma, unlike human tumors that also express COX-2 in the tumor cells. In general, these models have the limitation of having no measurable, COX-2-derived, prostaglandins, the inhibition of which correlates with antitumor efficacy. We report here that 1483 human head and neck xenograft tumors express COX-2 similar to the pattern observed in human solid tumors and that COX-2 activity produces high levels of prostaglandin E2 (PGE2). Inhibition of COX-2 by celecoxib resulted in loss of intratumor PGE2 levels and reduced tumor growth in a dose-dependent manner. In contrast, a selective COX-1 inhibitor, SC-560, did not measurably reduce tumor prostaglandin levels or tumor growth despite the presence of COX-1 in the host and tumor cells. Celecoxib-treated tumors showed reduced proliferation and increased apoptosis of both tumor and stromal cells compared with vehicle controls. Specific inhibition of PGE2 activity by a neutralizing antibody mimicked the reduced tumor growth observed after celecoxib treatment, suggesting growth is PGE2 mediated. These data indicate that a major antitumor mechanism of action of celecoxib is inhibition of COX-2-derived prostaglandins, particularly PGE2, and suggest celecoxib as a novel therapeutic agent for human head and neck cancer.

Cyclooxygenase-2 inhibition by celecoxib reduces proliferation and induces apoptosis in angiogenic endothelial cells in vivo.

Cyclooxygenase-2 (COX-2) is expressed within neovascular structures that support many human cancers. Inhibition of COX-2 by celecoxib delays tumor growth and metastasis in xenograft tumor models as well as suppresses basic fibroblast growth factor 2 (FGF-2)-induced neovascularization of the rodent cornea. The present studies were undertaken to evaluate possible mechanisms of the antiangiogenic and anticancer effects of celecoxib. Prostaglandin E(2) (PGE(2)) and thromboxane B(2) (TXB(2)) were increased in rat corneas implanted with slow-release pellets containing FGF-2 (338.6 ng of PGE(2)/g and 17.53 ng of TXB(2)/g) compared with normal rat corneas (63.1 ng of PGE(2)/g and 2.0 ng of TXB(2)/g). Celecoxib at 30 mg/kg/day p.o. inhibited angiogenesis (78.6%) and prostaglandin production by 78% for PGE(2) (72.65 ng/g) and 68% for TXB(2) (5.55 ng/g). Decreased prostaglandin production in corneas was associated with a 2.5-fold cellular increase in apoptosis and a 65% decrease in proliferation. Similar reductions in proliferation were observed in neovascular stroma (65-70%) of celecoxib-treated (dietary 160 ppm/day) xenograft tumors as well as in tumor cells (50-75%). Apoptosis was also increased in the tumor cells (2.2-3.0-fold) in response to celecoxib. Thus, the antitumor activity of celecoxib may be attributable, at least in part, to a direct effect on host stromal elements, such as the angiogenic vasculature.

The Role of Cyclooxygenase-2 in Inflammation.

Prostaglandins (PGs) can be synthetized via two isoforms of cyclooxygenase (COX). COX-1 is constitutively expressed in normal tissues, and its activity represent the normal physiological output of PGs. In inflammatory states, the newly discovered COX-2 is rapidly induced, and its activity accounts for the large amounts of PGs seen in inflammation. The commercially available nonsteroidal anti-inflammatory drugs (NSAIDs) are nonselective inhibitors of both COX isoforms; therefore, they provide anti-inflammatory activity as well as side effects associated with COX-1 inhibition. Selective inhibition of COX-2 expression explains at least in part the potent anti-inflammatory activity of steroids. Anti-inflammatory activity of newly developed COX-2 inhibitors, such as NS-398 or SC-58125, suggest a new approach of inflammatory diseases with more efficacious NSAIDs essentially devoid of side effects such as stomach ulcers.