Determination of a No-Observable Effect Level for Endotoxin Following a Single Intravitreal Administration to Dutch Belted Rabbits

Abstract

PURPOSE. The purpose of this study was to characterize the inflammatory response and determine a no-observable effect level (NOEL) in rabbit eyes after endotoxin intravitreal (ITV) injection.METHODS. Fifty-three naïve male Dutch Belted rabbits were treated with a single 50-lL ITV injection ranging from 0.01 to 0.75 endotoxin units/eye (EU/eye) and monitored for up to 42 days post treatment. Ophthalmic examination included slit-lamp biomicroscopy and indirect ophthalmoscopy. Laser flare photometry was performed in a subset of animals. On days 2, 8, 16, and 43, a subset of animals was necropsied and eyes processed for histopathological evaluation.RESULTS. Intravitreal injection of endotoxin at ‡0.05 EU/eye resulted in a dose-related anterior segment inflammation response. No aqueous flare or cell response was noted in the 0.01 EU/ eye dose group. A more delayed posterior segment response characterized by vitreal cell response was observed beginning on day 5, peaking on day 9, and decreasing starting on day 16 that persisted at trace to a level of 1þ on day 43. Microscopy findings of infiltrates of minimal mixed inflammatory cells in the vitreous and subconjunctiva and proteinaceous fluid in the anterior chamber and/or vitreous were observed in eyes given ‡0.1 EU/eye. CONCLUSIONS.We defined the NOEL for ITV endotoxin to be 0.01 EU/eye, suggesting that the vitreal cavity is more sensitive to the effects of endotoxin than the anterior segment and aqueous chamber. These data highlight the importance of assessing endotoxin level in intravitreal formulations, as levels as low as 0.05 EU/eye may confound the safety evaluations of intravitreal therapeutics in rabbits.

Systemic anti-inflammatory therapy aided by double-headed nanoparticles in a canine model of acute intraocular inflammation

“…A slit-lamp scoring system is semiobjective, based on the observer’s interpretation, and prone to bias; however, this method provides a more detailed and comprehensive picture of the inflammatory response of the eye by recording multiple parameters ( 29 ). In addition, quantitative measurements via photometry correlate well with clinical grades of aqueous flare when a standardized method of scoring is used ( 46 ). The SPOTS system was elected over other scoring systems as it provides enhanced applicability and validity of scoring criteria to canine species and incorporates ophthalmic findings commonly observed in preclinical studies involving ocular therapeutics ( 44 ).…”

Determination of a No Observable Effect Level for Endotoxin Following a Single Intravitreal Administration to Cynomolgus Monkeys

“…Recently, we characterized the sensitivity and time course of the response to endotoxin following ITV administration in rabbits. 16 Development of biologics, however, often requires the use of NHPs as the only pharmacologically relevant species. Thus, defining a no-observable-effect level (NOEL) for ITV endotoxin and characterization of the relationship of endotoxin dose to ocular inflammation in the NHP is important and will aid interpretation of ocular nonclinical safety data.…”

“…Using the same reference endotoxin material as the recently published ITV NOEL in rabbit, 16 the objective of this study was to characterize the acute ocular inflammatory response in a small number of animals and define the NOEL in the NHP to a range of endotoxin doses delivered by ITV injection.…”

Extended Intravitreal Rabbit Eye Residence of Nanoparticles Conjugated With Cationic Arginine Peptides for Intraocular Drug Delivery: In Vivo Imaging

“… 28 Manufacturing lipid NP is problematic because of their high affinity for endotoxin, 29 inserted in their structure, and the finding that tissue contacting vitreous is exceptionally sensitive to endotoxin. 30 Cationic particles, as described here, also attract anionic endotoxin micelles but, without surface lipid, these should be removable by immobilized poly-histidine. 31 …”