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A. Berra, F. M. Michelini, J. Ramirez, L. Galagovsky, L. Alche; In vitro and in vivo Anti-Herpetic and Anti-Inflammatory Activities of a New Synthetic Brassinosteroid Analogue. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5519.
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to evaluate the anti-herpetic and anti-inflammatory activities of a new molecule derived from brassinosteroids (32b) in a human conjunctival cell line (IOBA-NHC) and in an herpetic stromal keratitis (HSK) experimental model in mice.
In vitro: the 50% cytotoxic concentration (CC50) of 32b was determined by a MTT colorimetric assay in NHC cells. To establish the 50% effective concentration (EC50) of 32b, NHC cells were infected with Herpes simplex virus type 1 (HSV-1) KOS strain and supernatants were titrated in Vero cells. NHC cells infected with HSV-1 Cgal were fixed and stained for β-gal activity. In vivo: anaesthetized adult Balb/c mice were inoculated in the right cornea with 2.5 × 104 PFU of HSV-1. PBS (control), or acyclovir (ACV) 3%, or dexamethasone (DEX) 0.4% or 40µM 32b, were administered topically in the infected cornea, three times a day, following two schedules of treatment (1 to 3 days post-infection (p.i.) or 6 to 8 days p.i.), and signs of HSK were registered.
CC50 and EC50 values were 41.4 and 5.6 µM, with a selectivity index (CC50/ EC50) of 7.4. HSV-1 Cgal propagation was inhibited by 32b in NHC cells. At 15 days p.i., 75% of control mice showed signs of HSK, whereas ocular disease was evident in 30 %, 90 % and 40 % of mice treated at 1, 2 and 3 days p.i. with ACV, DEX and 32b, respectively. The severity of HSK in control mice (1.92 ± 0.25) and DEX (3.58 ± 0.38) was significantly higher than that of ACV (0.88 ± 0.43) and 32b (1.21 ± 0.35). When ACV, DEX and 32b were administered at 6, 7 and 8 days p.i., 100 %, 30 % and 40 % of mice, respectively, exhibited signs of HSK. The severity of disease in mice treated with DEX and 32b diminished considerably with respect to control and ACV-treated animals (1.18 ± 0.5 and 1.17 ± 0.36 vs. 1.92 ± 0.25 and 2.4 ± 0.41, respectively) Viral titres from eye washings significantly decreased only in ACV-treated mice.
Compound 32b displayed in vitro anti-herpetic activity and reduced the incidence and severity of HSK when administered under both schedules of treatment. Despite the lack of anti-HSV-1 activity in vivo, the fact that 32b improved the clinical signs of HSK leads us to consider that it may play a role in immune-mediated stromal inflammation. The mechanism of action of this compound would be different from that of DEX, since the administration of 32b at 1, 2 and 3 days p.i. did not exacerbate ocular damage as DEX did.
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