SCH 412499: biodistribution and safety of an adenovirus containing P21(WAF-1/CIP-1) following subconjunctival injection in Cynomolgus monkeys.

Monkey studies were conducted for the preclinical safety assessment of SCH 412499, an adenovirus encoding p21, administered by subconjunctival injection prior to trabeculectomy for postoperative maintenance of the surgical opening. Biodistribution of SCH 412499 was minimal and there was no systemic toxicity. Findings included swollen, partially closed or shut eye(s) and transient congestion in the conjunctiva. A mononuclear cell infiltrate was present in the conjunctiva, choroid and other ocular tissues, but completely or partially resolved over time. Electroretinograms and visual evoked potentials revealed no adverse findings. Thus, the findings are not expected to preclude the clinical investigation of SCH 412499.

Surgical lowering of elevated intraocular pressure in monkeys prevents progression of glaucomatous disease.

Recent reports from large clinical trials have clearly demonstrated that lowering intraocular pressure (IOP) in persons with ocular hypertension has a beneficial effect on reducing the progression of glaucomatous disease. Few studies of this effect have been conducted in controlled laboratory settings, however, none have been conducted using non-human primates, the model of experimental glaucoma considered most similar to the human disease. Using data collected retrospectively from a trabeculectomy study using 16 cynomolgous monkeys with experimental ocular hypertension, we evaluated both the threshold of elevated IOP required to cause clinically observable damage to the optic nerve head and also if lowering IOP below this threshold prevents further damage. An index of the level of elevated IOP experienced by experimental eyes (the Pressure Insult) was calculated as the slope of the difference in cumulative IOP between experimental and control eyes during four intervals of time over the course of the experiment, while damage to the optic nerve head was evaluated by measuring the Cup:Disc ratio for each eye from stereoscopic photographs taken at the end of each interval. An increase in the Cup:Disc ratio was significantly associated with both the maximum IOP obtained in the experimental eye during each interval (r=0.573, P<0.001) and the Pressure Insult (r=0.496, P<0.001). Pressure Insult values less than 11 mm Hg Days/Day were not associated with glaucomatous damage in monkey eyes, whereas values greater than 11 showed a significant correlation with increasing Cup:Disc ratios (P<0.001). Trabeculectomy to reduce the Pressure Insult below 11 was correlated with an attenuation of the rate of progression of the Cup:Disc ratio in eyes that had exhibited damage before surgery. These results contribute further to our understanding of this model of experimental glaucoma by demonstrating a threshold at which IOP needs to be elevated to stimulate damage, while also providing corroborating evidence that lowering IOP in ocular hypertensive monkeys can attenuate the progression of glaucomatous disease.

p21WAF-1/Cip-1 gene therapy as an adjunct to glaucoma filtration surgery.

Glaucoma is a blinding eye disease characterized by elevated intraocular pressure (IOP). Glaucoma filtration surgery (GFS) is designed to reduce IOP, but wound healing responses to the procedure can result in surgical failure. Anti-metabolites used in conjunction with GFS are commonly employed to control the wound healing response, but have unwanted side effects. This review describes the therapeutic potential of ocular gene therapy using an adenovirus vector containing the human p21WAF-1/Cip-1 gene (rAd-p21) to control unwanted wound healing post-GFS. Here, we summarize encouraging preclinical data in relevant models, and propose rAd-p21 gene therapy as an alternative to the currently used methods of wound healing modulation.

Involvement of tumor cell integrin alpha v beta 3 in hematogenous metastasis of human melanoma cells.

Early metastasis is the primary cause of death in melanoma patients. The adhesion receptor integrin alpha v beta 3 contributes to tumor cell functions that are potentially involved in melanoma growth and metastasis. We tested whether integrin alpha v beta 3 supports metastasis of human melanoma cells when injected into the bloodstream of immune deficient mice. Comparing variants of the same melanoma cell type that expressed either alpha v beta 3, alpha IIb beta 3 or no beta 3 integrin, we found that only alpha v beta 3 strongly supported metastasis. Inhibition of tumor cell alpha v beta 3 function reduced melanoma metastasis significantly and prolonged animal survival. To understand mechanisms that allow alpha v beta 3, but not alpha IIb beta 3 to support melanoma metastasis, we analyzed proteolytic and migratory activities of the melanoma cell variants. Melanoma cells expressing alpha v beta 3, but not those expressing alpha IIb beta 3 or no beta 3 integrin, produced the active form of metalloproteinase MMP-2 and expressed elevated mRNA levels of MT1-MMP and TIMP-2. This indicates an association between alpha v beta 3 expression and protease processing. Furthermore, alpha v beta 3 expression was required for efficient melanoma cell migration toward the matrix proteins fibronectin and vitronectin. The results suggest that expression of integrin alpha v beta 3 promotes the metastatic phenotype in human melanoma by supporting specific adhesive, invasive and migratory properties of the tumor cells and that the related integrin alpha IIb beta 3 cannot substitute for alpha v beta 3 in this respect.

Adenovirus-mediated gene therapy using human p21WAF-1/Cip-1 to prevent wound healing in a rabbit model of glaucoma filtration surgery.

OBJECTIVE: To determine if adenovirus-mediated p21(WAF-1/Cip-1) (p21) gene therapy can prevent fibroproliferation and wound healing in a rabbit model of glaucoma filtration surgery. METHODS: In vitro studies were performed using rabbit Tenon fibroblasts harvested from fresh tissue. In vivo studies were conducted in New Zealand white rabbits. A full-thickness sclerotomy was performed under a limbal-based conjunctival flap. Reagents tested included a replication-deficient recombinant adenovirus containing the human p21 gene (rAd.p21); the nonspecific marker gene for green fluorescent protein or beta-galactosidase; mitomycin, 0.5 mg/mL; and balanced saline solution. Each treatment was applied episclerally for 5 minutes before the sclerotomy using a soaked cellulose sponge placed under the surgically created conjunctival flap. Independent experiments were conducted to (1) monitor changes in intraocular pressure during a 30-day period after treatment and examine surgical site histological features, (2) examine changes in bleb morphologic features over 30 days, (3) determine outflow facility 14 days after treatment, and (4) examine the localization and persistence of rAd.p21 expression between 3 and 60 days after treatment. RESULTS: Treatment of Tenon fibroblasts with rAd.p21 resulted in a dose-dependent inhibition of DNA synthesis and cell growth in vitro. In vivo, rAd.p21 inhibited wound healing and fibroproliferation after filtration surgery, comparably to mitomycin. Mitomycin caused notable thinning of the bleb wall. In addition, 2 of the 5 mitomycin-treated eyes exhibited an abscess with hypopyon and hyalitis 30 days after surgery, which was not observed in any of the rAd.p21-treated eyes. None of the treatments resulted in a significantly sustained decrease in intraocular pressure during the 30-day period, although mitomycin treatment resulted in a significant (P =.02) increase in outflow facility 2 weeks after surgery in separate animals. Mitomycin- and rAd.p21-treated eyes had functioning blebs at the end of the experiment based on slitlamp examination. CONCLUSIONS: Mitomycin and rAd.p21 were effective in preventing fibroproliferation and wound healing in a rabbit model of glaucoma surgery. Mitomycin treatment increased outflow facility in normal-pressure eyes. CLINICAL RELEVANCE: Gene therapy with rAd.p21 may provide an effective antiproliferative for glaucoma filtration surgery, without the complications associated with mitomycin.