After establishing improved GSNOR potency some of the potent
After establishing improved GSNOR potency, some of the potent inhibitors were further evaluated for microsomal stability and CYP inhibition studies (). Majority of the tested analogs revealed high metabolic stability in human and rat liver microsomes, and moderate to low stability in mouse liver microsomes except few analogs (). CYP inhibition study of tetrazole analogs and revealed higher CYP inhibition (≥50% @ 10 µM) for all five major CYP isoforms (1A2, 2D6, 3A4, 2C9 and 2C19) tested. On the other hand, carboxylic PD128907 HCl australia analog unveiled minimal CYP inhibition to all five CYP isoforms evaluated. Likewise, 2-methylimidazole analog , which is structurally similar to lead provided gratifyingly low CYP inhibition for all the isoforms tested. In addition, substituted biphenyl derivatives , and exhibited higher CYP2C9 and CYP2C19 liability. Conversely, 2-methylimidazole analog , which is structurally similar to the potent analog still displayed CYP2C19 liability. Besides, moderate CYP2C9 and CYP2C19 liability were observed to pyridine derived biaryl compounds (, and ). Overall, compounds from this series still revealed either CYP2C9 or 2C19 and or both CYP isoform liabilities, which is undesirable. A single dose pharmacokinetic (PK) study of compounds and were carried out after intravenous () and oral administration on male C57 mice. As shown in , compound displayed low plasma clearance (CL) and volume of distribution (Vz) after 3 mg/kg dose in comparison to compound . As well, after 30 mg/kg PO administration, compound unveiled roughly 4-fold higher C and AUC in comparison to compound despite having similar oral half-life (T). The oral bioavailability of both compounds were found to be low to moderate (%F: 21 and 49). As GSNOR is expressed in lung, and COPD efficacy evaluation requires adequate lung distribution of orally available compounds, lung partitioning of and was evaluated and found to be comparable as disclosed in . Subsequently, compound was chosen for further profiling after having encouraging PK, metabolic stability and human GSNOR potency comparable to clinical lead compounds N6022 () and N91115 (). The mouse and rat potency of compound (mIC: 7.1 nM and rIC: 9.1 nM) were comparable to human GSNOR potency and further demonstrated good selectivity against alcohol dehydrogenase (ADH) and carbonyl reductase (CBR1) family of enzymes (). Compound appeared to be highly protein bound in human and mouse, and revealed low permeability in PAMPA assay and the corresponding efflux ratio was found to be low (0.8). However, CALU-3 (human airway epithelial cells) permeability of compound was found to be higher than PAMPA. The hERG channel activity of compound was found to be low (11% inhibition @10 µM in patch clamp) and further exhibited minimal off-target liability (few hits in CEREP panel data, which is not significantly relevant to GSNOR inhibitor). Solubility data of is also presented in both in Fed-state and fasted state and has low aqueous solubility. Consequently, compound was further evaluated for efficacy in cigarette smoke (CS) induced pulmonary inflammation for COPD in mice (widely used model for efficacy study)., Male C57BL/6 mice were exposed to Kentucky Research Grade cigarettes (3R4F) twice daily in a whole body inhalation exposure chamber (SIU24, ProMech Lab Holding AB, Sweden) for seven days, while GSNOR inhibitor was dosed orally for seven days, one hour prior to 1st smoke exposure. The PDE4 inhibitor, roflumilast (approved oral drug) was used as a positive control, naive mice exposed to air (vehicle) was served as negative control. On day-8 after the last smoke exposure, animals were euthanized and subjected to bronchoalveolar lavage (BAL), and then fluid was collected. Total and differential BAL cell counts were performed from the BAL fluid. Data was analyzed using Graph Pad Prism software and statistical analysis was conducted using one way ANOVA followed by Dunnett’s multiple comparison test. Study results revealed significant dose dependent inhibition of cellular infiltration in BALF of the animals exposed to cigarette smoke upon compound treatment, and the efficacy dose of 100 mg was comparable to Roflumilast (). Thus, lead compound (oral GSNOR inhibitor) demonstrated significant anti-inflammatory effects in cigarette smoke induced pulmonary inflammation in mice.