Peter J. Kelleher

Inhibition of cell proliferation on lens capsules by 4197X-ricin A immunoconjugate+++

Purpose: To evaluate the cytotoxicity of immunotoxin 4197X‐ricin A (4197X‐RA) and its ability to inhibit protein synthesis and human lens epithelial cell (LEC) proliferation on the inner surface of the lens capsule. Setting: Houston Biotechnology, Inc., The Woodlands, Texas. Methods: A cell culture system was established using human LECs as a model for the proliferation of remnant LECs that occurs during posterior capsule opacification (PCO) after extracapsular cataract extraction. The LEC culture system was also used in vitro for testing compounds that might inhibit this process in vivo. Human LECs were cultured on the surface of the original lens capsule fixed to collagen. Variability was reduced by dissecting each lens capsule into equivalent halves and exposing the segments to immunotoxin 4197X‐RA. Results: Protein synthesis and LEC proliferation were almost completely inhibited at relatively low 4197X‐RA concentrations after short exposure. The inhibitory effects persisted up to 3 weeks after withdrawal of the immunotoxin and after several media exchanges. Conclusion: Immunotoxin 4197X‐RA may help prevent PCO after primary cataract surgery.

Effects of daunomycin implants on filtering surgery outcomes in rabbits

PURPOSEnTo evaluate the effects of a novel daunomycin (DM) implant on intraocular pressure (IOP), bleb survival, and anterior segment complications when administered during filtering surgery on New Zealand White rabbits.nnnMETHODSnImplants were prepared by covalent coupling of DM to a high molecular weight hyaluronic acid (HA) and fabricated into solid implants containing 250, 65, and 25 microg of DM. Full thickness sclerostomies were performed, and rabbits received no implant (control), placebo implant containing HA, or an implant containing HA-DM conjugate at the time of surgery. Rabbits were then followed for 30 days to assess change in IOP, bleb survival, and anterior segment associated complications.nnnRESULTSnIn vitro, the release of DM from the implants was a first order process with a half-life of 51 h. In control rabbits, rabbits receiving placebo implant, and rabbits receiving HADM (250 microg) implants, the mean IOPs on day 3 were 11.1 +/-1.6, 10.8 +/- 2.7, and 14 +/- 0.98 mm of Hg, respectively. On days 5 through 9, IOP in the control and placebo-implanted groups returned to preoperative levels. However, in rabbits receiving 250 microg of conjugated DM, mean IOP on day 7 was reduced from preoperative levels by 11.8 +/- 3.2 mm of Hg (P < 0.05). This reduction in IOP was significantly different (P < 0.05) from both control and placebo implant groups, and IOPs remained at these levels until studies were terminated on day 30. In control and placebo-implanted rabbits, bleb size started decreasing on day 1, and by day 7, no blebs were observed. In rabbits receiving 250 microg HA-DM implants, mean bleb survival time was greater than 30 days. The DM-induced reduction in IOP and enhanced bleb survival was dose-dependent. In rabbits receiving 65 microg of conjugated DM, lOPs were significantly reduced through day 30; however, at times beyond day 19 there was a gradual rise in mean IOP as filtering procedures in individual animals began to fail. Mean bleb survival time was greater than 30 days for implants containing 65 microg of conjugated DM. In rabbits receiving HA-DM implant containing 25 microg of DM, IOPs were not significantly different from preoperative levels beyond day 9, and no significant enhancement in bleb survival was observed in these animals. Comparison of HA-DM conjugated implants to those containing equal doses of free DM demonstrated that the mean IOP change at 30 days were similar; however, there was a reduction in anterior segment complications associated with the use of HA-DM conjugated implants.nnnCONCLUSIONSnThis study provides evidence that the controlled release of DM when conjugated to HA can significantly improve the success of filtering procedures. The maintenance of ocular hypotension and bleb survival along with the reduction in anterior segment complications supports the idea that HA-DM conjugate will be more efficacious than the use of free DM in improving the success rate of filtering surgery.

Ocular pharmacokinetics of lens epithelial cell-specific immunotoxin 4197X-RA

The ocular clearance, stability and systemic distribution of a lens epithelial cell-specific immunotoxin were evaluated in adult Dutch Belted rabbits. Immunotoxin 4197X-RA was prepared from a murine monoclonal antibody conjugated by a disulfide linkage to ricin A. After intracameral (i.c.) injection, clearance of the immunotoxin from the eye followed a first-order process. The half-life (t1/2) of immunotoxin in the aqueous humor was 51 min. Accumulation of the immunotoxin in ocular tissues appeared to be associated with aqueous outflow tracts, as it was primarily confined to the iris and the limbal regions of the cornea. No acute metabolism of immunotoxin was observed in the anterior chamber. The immunotoxin was detected in the blood 20 min post-injection and achieved a steady-state level after 40 to 180 min. A slow decline in labeled protein was then observed from 180 min to 24 hr. Systemic clearance of the immunotoxin appeared to occur primarily by way of the kidneys. The present data indicate that immunotoxin 4197X-RA, like other large proteins, is passively cleared from the anterior chamber by aqueous flow, and that little or no acute breakdown occurs in the anterior chamber. It is anticipated that these data, along with results from cytotoxicity studies, will result in the determination of optimal doses and frequency of administration for the use of immunotoxin in human clinical trials.