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Introduction: This study investigated the efficacy of pooled human immune globulins (Flebogamma® DIF) to combat the formation of neutrophil extracellular traps (NETs) and NETosis, along with neutrophil adhesion to corneal epithelial cells in response to dry eye disease relevant stimuli. Methods: Human neutrophils were isolated by bead-based immunomagnetic depletion of non-target cells from human whole blood. NETosis was induced using phorbol 12-myristate 13-acetate (PMA) or anti-citrullinated histone 4 R3 antibody (H4R3 ACPA). Extracellular DNA was used as a surrogate biomarker of NETosis, and it was quantified using a 96-well, plate reader-based fluorescent assay and by confocal microscopy in 8-well chambers using the DNA dye, SYTOXTM Green. Neutrophils were labeled with calcein-AM and adhesion to human corneal epithelial cells was measured. The efficacy of a dose-range of pooled human immune globulin (Flebogamma® DIF, 0.01%–5%) was tested in all assays. Results: Pooled human immune globulins (Flebogamma® DIF) dose-dependently inhibited both PMA and H4R3 ACPA induced NETosis, with concentrations ≥2.5% fully preventing release of extracellular DNA over a 2–16 h time period. Similarly, Flebogamma® 5% DIF prevented NETosis against PMA (20 nM) and a dose range (0.1–10 μg/mL) of H4R3 ACPA. Both PMA and H4R3 ACPA increased adhesion of neutrophils to corneal epithelial cells by 20% and 5%, respectively. Flebogamma® DIF treatment resulted in a dose-dependent reduction of neutrophil adhesion, with Flebogamma® 5% DIF reducing adhesion to baseline levels. Discussion: These findings show the dose-dependent efficacy of pooled human immune globulins, specifically Flebogamma® DIF against experimentally and pathologically induced NETosis and neutrophil adhesion to corneal epithelial cells, in vitro. The results from this study support the continued clinical development of Flebogamma® 5% DIF as a novel and efficacious treatment for the signs and symptoms of dry eye disease. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the safety and tolerability of pooled human immune globulins, Flebogamma® 5% DIF and Flebogamma® 10% DIF, administered by topical ophthalmic instillation to New Zealand White (NZW) rabbits. Methods: Male NZW rabbits were used in this study. In the acute single dose tolerability study, rabbits (n = 12) received a single topical dose of Flebogamma® 5% DIF. In the two-week repeated-dose tolerability study, rabbits (n = 5 for each group) were administered either Flebogamma® 5% DIF or Flebogamma® 10% DIF by topical bilateral administration four times daily (q.i.d.) between 8 am and 6 pm for a period of two weeks. Full ophthalmic examinations were conducted to evaluate ocular tolerability at baseline, Day 7, and Day 14. Results: In the acute single dose study, mild hyperaemia was observed in 1 out of 4 eyes at each 4 h and 24 h post-instillation of Flebogamma® 5% DIF. In the repeated dose study, no ocular signs were detected after q.i.d. topical instillation of Flebogamma® 5% DIF, while Flebogamma® 10% DIF resulted in mild hyperaemia in 8 out of 10 eyes on Day 7, and 5 out of 10 eyes on Day 14. No positive corneal fluorescein staining was detected. Schirmer tear test results were unremarkable. No other ocular signs were observed. Administration of immune globulins had no effect on intraocular pressure. Conclusions: Flebogamma® 5% DIF and Flebogamma® 10% DIF were well-tolerated by NZW rabbits following single and repeat dose topical ophthalmic administration, supporting the future development of topical pooled human immune globulins for the treatment of ocular surface disease. [ABSTRACT FROM AUTHOR]