Quantization of viral DNA was performed using real-time quantitative PCR and using the standard curve generated in parallel and expressed as a concentration of ng/μl. (D) The viral DNA was extracted from Vero cells 24h post HSV-1 infection and NAABs treatment (0.06mg/mL), as described in the Materials and Methods. The cells were stained with crystal violet for plaques detection. After the incubation time, the inoculum was removed, and the monolayers were overlaid with DMEM containing 0.8% methylcellulose in the presence of Acyclovir at 0.5, 1, 10, and 20 μM. (C) Antiviral activity of Acyclovir: Vero cells infected with HSV-1 (F) and incubated for 1 h at 37 ☌. The EC 50 was obtained from nonlinear regression analysis of concentration-effect curves by the GraphPad Prism 6 program.
(B) Antiviral activity of NAAB-496 and NAAB-503 was evaluated on the basis of plaques reduction assay in Vero cells infected with HSV-1 (F) and incubated in the presence of NAAB 496 and NAAB 503 at different concentrations (0.01, 0.05, 0.06, 0.08 and 0.1 mg/mL).
The CC 50 was obtained from nonlinear regression analysis of concentration-effect curves by the GraphPad Prism 6 Demo program and expressed the means ± standard deviation of three independent experiments. The cell proliferation index (%) was determined at 72 hours of treatment on the basis of ATP levels using the ViaLight Plus Cell Proliferation and Cytotoxicity BioAssay Kit (Lonza Group Ltd., Basel, Switzerland). (A) The cytotoxicity effect of NAABs was evaluated on Vero cells in presence of different concentrations of NAABs compound (0.01, 0.05, 0.06, 0.08, 0.1, 0.15, 0.2, 0.4, 0.6, 0.8 and 1.6 mg/mL) separately. Viability assay and Antiviral activity of NAAB-496 and NAAB-503 in Vero cells. Kinetic of degradation of NAAB-496 and NAAB-503 after gastric (A) and gastric plus duodenal digestion (B). Method validation parameters of NAAB-503 and NAAB-496.
Representative chromatograms of NAAB-503 (A) degradation after gastric (B) and gastric plus duodenal digestion (C). Representative chromatograms of NAAB-496 (A) degradation after gastric (B) and gastric plus duodenal digestion (C). Representative chromatograms of NAAB-496 and NAAB-503 reference standard solutions (10 μg/mL). Chemical structures of compounds NAAB-496 and NAAB-503.
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