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.

Keywords: drug development; endotoxin; intravitreal injection; ocular toxicology.

FIG. 1.

On day 1, IOP spiked immediately following the first injection, as expected due to bolus administration. Ten minutes after the first injection (before the second injection), IOP returned to pre-ITV injection baseline values. IOP spike was also noted following the second injection, as expected and 10 min afterward it returned to baseline values. IOP, intraocular pressure; ITV, intravitreal. Color images are available online.

FIG. 2.

Following ITV endotoxin administration, a rapid, transient aqueous inflammatory response was observed. Dose-dependent aqueous flare was observed at ≥0.04 EU/eye (A; results expressed as individual eye measurements). This response peaked approximately on day 2 and reached an average of Grade 3–4+ in eyes given ≥0.2 EU/eye. Aqueous flare resolved by day 5 in a subset of animals given 0.04 EU/eye, and by day 15 in eyes given ≥0.2 EU/eye. Aqueous flare was not noted in vehicle-treated eyes or eyes given 0.01 EU/eye. The aqueous cell response (B) followed a similar dose-related trend. Aqueous flare was not noted in vehicle-treated eyes or eyes given 0.01 EU/eye. EU, endotoxin units. Color images are available online.

FIG. 3.

The vitreal haze was observed at doses >0.01 EU/eye (A). In general vitreous haze scores were dose-related (A; results expressed as individual eye measurements). Vitreal cell response was first observed on day 2 at low levels and gradually increased in a roughly dose-associated manner through day 8 and then plateauing or increasing slightly after day 8 (B; results expressed as individual eye measurements). Vitreous haze or cell was not noted in vehicle-treated eyes or eyes given 0.01 EU/eye. Color images are available online.

FIG. 4.

Representative color fundus images of the right eye of an animal that had an ITV injection of 0.35 EU/eye endotoxin are shown here. (A) Baseline; (B) Day 8; and (C) Day 15. Hazy media and optic nerve swelling are seen on day 8 (B), both of which had partially resolved on day 15 (C). Color images are available online.

FIG. 5.

OCT images from animals given Endotoxin at 0.21 EU/eye and 0.35 EU/eye showed RNFL thickening (white arrows) compared to baseline (A, B), an increase in white hyper reflective spots in the vitreous (white arrows) (C), ILM wrinkling (white arrows) (D). Anterior segment scans on day 2 showed an increase in reflective spots and clumps (white arrows) that could represent cells and fibrin (E). All of these effects are consistent with an inflammatory response noted on clinical ophthalmic examination. ILM, inner limiting membrane; OCT, optical coherence tomography; RNFL, retinal nerve fiber layer. Color images are available online.

FIG. 6.

Microscopic findings of minimal mononuclear cell infiltrates in the vitreous at ≥0.21 EU/eye were observed on day 15. Infiltrates occasionally extended into the optic disc (inset). Color images are available online.