In vitro and in vivo Anti-Adenovirus and Anti-Inflammatory Activities of a New Synthetic Brassinosteroid Analogue

Abstract

Purpose: : to evaluate the anti-adenovirus and anti-inflammatory activities of a new molecule derived from brassinosteroids (32b) in a human conjunctival cell line (IOBA-NHC) and in an adenoviral queratoconjunctivitis (ADV-QC)) experimental model in rabbit

Methods: : In vitro: the 50% cytotoxic concentration (CC₅₀) of 32b was determined by a MTT colorimetric assay in IOBA-NHC cells. To establish the 50% effective concentration (EC₅₀) of 32b, NHC cells were infected with adenovirus strain 5 (ADV-5) and supernatants were titrated in A549 monolayer cells. IOBA-NHC cells infected with ADV were treated with 4 concentration of 32b compound (2µM, 5 µM, 10 µM and 20 µM). TNF, NF-kB and viral titration were evaluated. In vivo: Adult New Zealand rabbit (NZ) were anesthetized. The central cornea was inoculated intrastromally to form 5 focal bleds (50 µl of ADV-5, (3.5 x 10⁸ pfu/ml)), scarified and by topically applying an additional 50 µl of ADV-5. Three concentration of 32b (10µM, 50 µM, and 100 µM) were administered topically in uninfected and infected cornea, three times a day, following schedule of treatment (1 to 21 days post-infection (p.i.) and signs ocular toxicity and ADV-QC were registered, respectively

Results: : In vitro: CC₅₀ and EC₅₀ values were 63.2 and 2 µM, with a selectivity index (CC₅₀/ EC₅₀) of 31.6 ADV propagation was inhibited by 32b in NHC cells. Compound 32b blocked ADV-5-induced activation of NF-ΚB by inhibiting its translocation to the nucleus of infected conjunctival cells in vitro. 32b significantly reduced the secretion of TNF- in a macrophage cell line. In vivo: 32b compound ((10µM, 50 µM, and 100 µM) showed no signs of ocular cytotoxicity. Infected rabbits without treatment developed acute conjunctivitis 1-8 pi, blefaroconjunctivitis and corneal edema (9-21) and infiltrating immune from day 15th p.i. The severity of disease (acute conjunctivitis, blefaroconjunctivitis and corneal edema and non showed sign of immune corneal infiltrate) in rabbit treated with 32b (100 µM) diminished considerably with respect to control infected animals.

Conclusions: : Compound 32b displayed in vitro anti-adenovirus and anti-inflammatory activities and in vivo reduced the incidence and severity of ADV-QC when administered under schedule of treatment. This data suggests that 32b could be a new drug in epidemic adenoviral queratoconjunctivitis

Keywords: adenovirus • keratitis • pathology: experimental