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

Abstract

Purpose: To characterize the inflammatory response and determine the no-observable-effect level (NOEL) in cynomolgus monkey eyes after intravitreal (ITV) injection of endotoxin. Methods: The inflammatory response to endotoxin was assessed in a single-dose study in monkeys at doses of 0.01 to 0.51 endotoxin units (EU)/eye. Tolerability was assessed by clinical ophthalmic examinations, intraocular pressure measurements, fundus color photography, optical coherence tomography, and anatomic pathology. Results: ITV injection of endotoxin at ‡0.04 EU/eye resulted in a dose-related anterior segment inflammatory response. No aqueous flare or cell was noted in the 0.01 EU/eye dose group. A more delayed posterior segment response characterized by vitreous cell was observed beginning on day 5, peaking on day 15, and decreasing in some groups. Microscopic findings of mononuclear cell infiltrates in the vitreous were observed in eyes given ‡0.21 EU/eye. Conclusion:The NOEL for ITV endotoxin in cynomolgus monkeys was 0.01 EU/eye, suggesting that this species is as sensitive as rabbits to the effects of endotoxin. The vitreous cavity also appears more sensitive to endotoxin than the anterior segment/aqueous chamber. Overall, the magnitude of the inflammatory response at ‡0.04 EU/eye suggests that dose-response curve in monkeys is steeper than in rabbits. These data highlight the importance of assessing endotoxin level in ITV formulations, as levels as low as 0.04 EU/eye may confound the safety evaluations of ITV therapeutics in cynomolgus monkeys.

Evaluation of the Dose-Dependent Inflammatory Response and No-Observable Adverse Effect Level of Intravitreal Endotoxin in the African Green Monkey

“…Results reported in the cynomolgus macaque included mild microscopic findings that were detected in eyes dosed with ≥0.21 EU and collected on day 15, a time point after resolution of most clinical examination findings. 5 We detected similar microscopic changes at the lower level of 0.04 EU in eyes collected on day 2 at the peak of inflammation following endotoxin administration. It may have been the case that, had eyes been collected at peak inflammation in the cynomolgus macaque study, microscopic changes would have been detected at a lower dose, similar to our findings in the green monkey.…”

“…Although acknowledging technical and methodological differences among the study designs, these findings confirm our hypothesis that green monkeys are as sensitive to intravitreal endotoxin as cynomolgus macaques and rabbits. 4 , 5 …”

“…Three reported studies have evaluated the effect of intravitreal endotoxin, quantified in endotoxin units (EU), in animal model species, including two in rabbits and a single study in cynomolgus macaques. 3 , 4 , 5 In rabbits, a NOAEL was identified as 0.01 EU/eye following evaluation of the dose range from 0.01 to 0.75 EU/eye. Dose-related inflammation was identified beginning at 0.05 EU/eye, with histopathological changes identified at 0.1 EU/eye.…”

“…Dose-related inflammation was identified in the cynomolgus monkeys beginning at 0.04 EU/eye, and histopathological changes were seen at 0.21 EU/eye. 5 These studies provide valuable comparative biology references for evaluation of the NOAEL in other preclinical species and humans.…”

Pharmacokinetics of Pullulan–Dexamethasone Conjugates in Retinal Drug Delivery

“…All tested concentrations (2.5, 1.25 and 0.63 mg/mL) of fluorescently labelled BDP-pullulan-DEX showed endotoxin levels below 0.03 mEU/µL. Thus, endotoxin levels in the formulations used for in vivo administration are at acceptable levels below 0.2 EU per injection [46,47].…”