Rapid Development of Glaucoma Via ITV Nonselective ANGPT 1/2 Antibody: A Potential Role for ANGPT/TIE2 Signaling in Primate Aqueous Humor Outflow.

Purpose: Investigate a significant, dose-related increase in IOP, leading to glaucomatous damage to the neuroretina and optic nerve following intravitreal (ITV) administration of a bispecific F(ab')2 [anti-VEGF/Angiopoietins [ANGPT]F(ab')2] molecule in adult monkeys. Methods: ITV ocular tolerability and investigation of anti-VEGF/ANGPT F(ab')2 (blocking both ANGPT1 and ANGPT2) was done in monkeys; mechanistic studies were done in neonatal mice. Results: Following the second ITV dose of anti-VEGF/ANGPT F(ab')2, all 1.5- and 4-mg/eye treated monkeys developed elevated IOP, which eventually was associated with optic disc cupping and thinning of the neuroretinal rim. Histopathologic examination showed nonreversible axonal degeneration in the optic nerves of animals administered 1.5 mg/eye and higher that was considered secondary to high IOP. Anti-ANGPT Fab also caused elevated IOP in monkeys, but anti-VEGF Fab did not contribute to the IOP increase. In addition, an anti-ANGPT2-selective antibody did not change IOP. In mice simultaneous blockade of ANGPT1 and ANGPT2 impaired the expansion and formation of Schlemm's canal (SC) vessels, similar to genetic ablation of Angpt1/Angpt2 and their receptor TIE2. As previously reported, blocking ANGPT2 alone did not affect SC formation in mice. Conclusions: Dual inhibition of ANGPT1/ANGPT2, but not ANGPT2 alone, leads to increased IOP and glaucomatous damage in monkeys. This confirms a role for TIE2/ANGPT signaling in the control of IOP in adults, a finding initially identified in transgenic mice. Dual pharmacologic inhibition of ANGPT1/ANGPT2 may affect aqueous drainage and homeostasis in adult monkeys and may be useful in developing novel models of glaucoma.

Glaucoma Drugs in the Pipeline.

Glaucoma is a chronic disease that can be challenging to treat for both patients and physicians. Most patients will require more than 1 medication over time to maintain their intraocular pressure (IOP) at a physiologically benign level. Patients may become refractory to existing compounds and many struggle with adherence to multiple topical drop regimens. The field of glaucoma therapeutics has been advancing rapidly with an emphasis on compounds comprising multiple molecules/mechanisms of action that offer additivity and are complementary to current therapeutics. Several new topical drop compounds directly targeting the trabecular meshwork (TM)/Schlemm canal/conventional outflow pathway to reduce outflow resistance have obtained US Food and Drug Administration approval in the past year. These include rho kinase inhibitors and nitric oxide donating compounds. Alternative therapies that offer long-term IOP lowering while removing the patient from the drug delivery system are moving forward in development. These include gene therapy and stem cell strategies, which could ease or eliminate the burden of topical drop self-administration for several years. Additionally, a variety of novel formulations and devices are in development that aim for controlled, steady state delivery of therapeutics over periods of months. The future of glaucoma therapy is focusing on an increase in specificity for the individual patient: their type of glaucoma; underlying mechanisms; genetic make-up; comorbid conditions; and rate of progression. Maintaining functional vision and improving patient outcomes remains the goal in glaucoma therapeutics. The current collection of novel therapeutics offers an expanded set of tools to achieve that goal.

Exciting directions in glaucoma.

Glaucoma is a complex, life-long disease that requires an individualized, multifaceted approach to treatment. Most patients will be started on topical ocular hypotensive eyedrop therapy, and over time multiple classes of drugs will be needed to control their intraocular pressure. The search for drugs with novel mechanisms of action, to treat those who do not achieve adequate intraocular pressure control with, or become refractory to, current therapeutics, is ongoing, as is the search for more efficient, targeted drug delivery methods. Gene-transfer and stem-cell applications for glaucoma therapeutics are moving forward. Advances in imaging technologies improve our understanding of glaucoma pathophysiology and enable more refined patient evaluation and monitoring, improving patient outcomes.

Application of canaloplasty in glaucoma gene therapy: where are we?

PURPOSE: Schlemm's canal (SC) inner wall is adjacent to the juxtacanalicular trabecular meshwork (TM) over their entire circumference. We seek to transfer reporter and therapeutic genes to these outflow-modulating tissues via canaloplasty surgery in live monkeys. METHODS: A standard canaloplasty surgical approach was performed in cynomolgus monkeys using flexible canaloplasty catheters, modified for monkey eyes with a 175-µm outer diameter and an LED-lighted tip. A 6-0 prolene suture was used for the exact localization of SC. Trypan blue was injected during catheter withdrawal to document catheter placement within SC and to determine ease of injecting fluid into SC. Before, during, and after the injection, the position of the catheter and the anatomic details were video-captured with an externally positioned noncontact endoscopic imaging system and 50 mHz ultrasound biomicroscopy (UBM). RESULTS: A 360° catheterization and injection of dye into SC was achieved. Suture, catheter, and trypan blue were imaged with the endoscope camera system and the catheter was also visualized with UBM. Trypan blue was seen in the SC over 5 clock hours after a 1 clock-hour insertion of the catheter. CONCLUSIONS: A modified canaloplasty catheter device might be used for gene delivery to the SC/TM area without circumferential catheterization. Further studies comparing different delivery methods of the vector/transgene into the SC using canaloplasty are needed.

Self-complementary AAV virus (scAAV) safe and long-term gene transfer in the trabecular meshwork of living rats and monkeys.

PURPOSE: AAV vectors produce stable transgene expression and elicit low immune response in many tissues. AAVs have been the vectors of choice for gene therapy for the eye, in particular the retina. scAAVs are modified AAVs that bypass the required second-strand DNA synthesis to achieve transcription of the transgene. The goal was to investigate the ability of AAV vectors to induce long-term, safe delivery of transgenes to the trabecular meshwork of living animals. METHODS: Single doses of AAV2.GFP and AAV2.RGD.GFP/Ad5.LacZ were injected intracamerally (IC) into rats (n = 28 eyes). A single dose of scAAV.GFP was IC-injected into rats (n = 72 eyes) and cynomolgus monkeys (n = 3). GFP expression was evaluated by fluorescence, immunohistochemistry, and noninvasive gonioscopy. Intraocular pressure (IOP) was measured with calibrated tonometer (rats) and Goldmann tonometer (monkeys). Differential expression of scAAV-infected human trabecular meshwork cells (HTM) was determined by microarrays. Humoral and cell-mediated immune responses were evaluated by ELISA and peripheral blood proliferation assays. RESULTS: No GFP transduction was observed on the anterior segment tissues of AAV-injected rats up to 27 days after injection. In contrast, scAAV2 transduced the trabecular meshwork very efficiently, with a fast onset (4 days). Eyes remained clear and no adverse effects were observed. Transgene expression lasted >3.5 months in rats and >2.35 years in monkeys. CONCLUSIONS: The scAAV viral vector provides prolonged and safe transduction in the trabecular meshwork of rats and monkeys. The stable expression and safe properties of this vector could facilitate the development of trabecular meshwork drugs for gene therapy for glaucoma.

Bioinformatic and statistical analysis of the optic nerve head in a primate model of ocular hypertension.

BACKGROUND: The nonhuman primate model of glaucomatous optic neuropathy most faithfully reproduces the human disease. We used high-density oligonucleotide arrays to investigate whole genome transcriptional changes occurring at the optic nerve head during primate experimental glaucoma. RESULTS: Laser scarification of the trabecular meshwork of cynomolgus macaques produced elevated intraocular pressure that was monitored over time and led to varying degrees of damage in different samples. The macaques were examined clinically before enucleation and the myelinated optic nerves were processed post-mortem to determine the degree of neuronal loss. Global gene expression was examined in dissected optic nerve heads with Affymetrix GeneChip microarrays. We validated a subset of differentially expressed genes using qRT-PCR, immunohistochemistry, and immuno-enriched astrocytes from healthy and glaucomatous human donors. These genes have previously defined roles in axonal outgrowth, immune response, cell motility, neuroprotection, and extracellular matrix remodeling. CONCLUSION: Our findings show that glaucoma is associated with increased expression of genes that mediate axonal outgrowth, immune response, cell motility, neuroprotection, and ECM remodeling. These studies also reveal that, as glaucoma progresses, retinal ganglion cell axons may make a regenerative attempt to restore lost nerve cell contact.

Ocular drug delivery: molecules, cells, and genes.

Recent advances in molecular cell biology have led to the exploration of new therapies based on molecules, cells, and genes to treat a variety of ocular disorders. In this review, we present the current state of knowledge pertaining to the development of different delivery systems to mediate safe and long-lived therapies, with an emphasis on gene therapy. The advantages and limitations of these delivery and therapeutic methods are also discussed.

Viscocanalostomy in rhesus monkeys.

OBJECTIVE: To examine structural changes and aqueous humor outflow after viscocanalostomy in live normal monkey eyes. METHODS: Viscocanalostomy surgery was performed in 1 eye of each of 4 rhesus monkeys. Outflow facility was determined before and after surgery. All eyes were fixed and examined by light and/or electron microscopy 36 or 63 days postoperatively. RESULTS: Schlemm canal was replaced by scar tissue at the surgical site. The juxtacanalicular zone contained homogeneous material, probably high-molecular-weight 1.4% sodium hyaluronate. The sclera external to Schlemm canal was overhydrated, and remains of a scleral lake were present in 1 animal. Multiple defects were present in the endothelial lining of Schlemm canal inner and outer wall. Fine fibrillar material and sheath-derived plaque material partly bridged the defects. Along the inner wall, aggregations of thrombocytes covered some defects in the endothelial lining of the canal. At 90 degrees to 180 degrees from the surgical site, small and fewer breaks in the inner wall were seen. Postsurgery outflow facility (n = 2) was approximately 30% higher in the treated eye than in the contralateral control, corrected bilaterally for presurgery baseline. CONCLUSIONS: The most likely explanations for the increase in outflow facility in monkeys after viscocanalostomy are focal disruptions of the inner wall endothelium of Schlemm canal and disorganization of the juxtacanalicular zone, resulting in direct communication of juxtacanalicular zone extracellular spaces with the lumen of Schlemm canal. The continuous presence of sodium hyaluronate might prevent repair of these defects by interfering with thrombocyte function. CLINICAL RELEVANCE: In nonhuman primates, viscocanalostomy appears to decrease outflow resistance through persisting focal disruption of the inner wall endothelium and opening of the juxtacanalicular or cribriform region of the trabecular meshwork, the tissue most affected by pathologic changes in primary open-angle glaucoma in humans.