Sildenafil citrate

PDE5A1 activity was assayed, as described earlier, with minor modification [24 (link)]. Briefly, PDE5A1 (75.3 µg/mL) prepared in Tris–HCl (50 mM; pH = 8.0), MgCl₂ (10 mM) and DTT (1 mM) was mixed with 10–30 nM of ³H-cGMP (20,000–30,000 cpm/assay, GE Healthcare, Buckinghamshire, UK) as a substrate as well as pGlu (Sigma-Aldrich, Berlin, Germany; purity ≥99%) and sildenafil citrate (Sigma Aldrich, Berlin, Germany; European Pharmacopoeia (EP) reference standard) as inhibitors (at concentrations ranging from 3 to 10 µM). The enzymatic reaction took place at 25 °C for 20 min and then the reaction was stopped by adding ZnSO₄ (0.2 N). Further, the reactant ³H-GMP was accelerated by Ba(OH)₂ (0.2 N), while unreacted ³H-cGMP was kept in the supernatant. PerkinElmer 2910 liquid scintillation counter (Thermo Fisher Scientific, Paisley, UK) was utilized to measure the radioactivity of the supernatant through 2.5 ml Ultima Gold liquid scintillation cocktails (Thermo Fisher Scientific, Paisley, UK). The half-maximal inhibitory concentration (IC₅₀) values for test inhibitors were ascertained in the presence of proper concentrations of ³H-cGMP (10–30 nM) and the enzyme (75.3 µg/mL) that hydrolyze up to 70% of the substrate and subsequently calculated by nonlinear regression analysis. Each measurement was conducted in triplicate.