In vitro activity: [3H]Aclidinium binds to a homogeneous receptor of M2 with Kd of 0.34 nM and Bmax of 3.13 pmol/mg, and binds to M3 with Kd of 0.34 nM and Bmax of 3.13 pmol/mg. Aclidinium (< 100 nM) dose-dependently inhibits carbachol-induced contractions in isolated guinea pig trachea. Aclidinium shows an onset of action with t1/2 of 6.8 min, tmax of 35.9 min in isolated guinea pig trachea. Aclidinium is hydrolysed in plasma samples from all species studied, with apparent half-lives at 37℃ of 11.7 min, 38.3 min, 1.8 min and 2.4 min in rat, guinea pig, dog and human plasma, respectively. Aclidinium (0.1 μM) inhibits carbachol and TGF-β1 induced upregulation of collagen type I and α-SMA mRNA and protein expression in human bronchial fibroblasts. Aclidinium (0.1 μM) inhibits TGF-β1 induced upregulation of ChAT expression in human bronchial fibroblasts. Aclidinium (0.1 μM) inhibits carbachol- and TGF-β1-induced increases in ERK1/2 phosphorylation and RhoA-GTP formation in human bronchial fibroblasts. Aclidinium pretreatment prevents the upregulation of M1 and M3, but not M2 downregulation induced by carbachol or TGF-β1 in human lung fibroblasts. Aclidinium (0.1 μM) dose-dependently inhibits the TGF-β1 and carbachol-induced cell proliferation of human lung fibroblasts.

Kinase Assay: The affinity of Aclidinium for the different human muscarinic receptor subtypes at equilibrium is determined by measuring their ability to displace the binding of [3H]NMS to cell membrane preparations expressing one of the human muscarinic receptor subtypes. Protein concentrations are 8.1 μg/well, 10.0 μg/well, 4.9 μg/well, 4.5 μg/well, and 5.0 μg/well for M1, M2, M3, M4, and M5 receptor membrane preparations, respectively. The assays are conducted at [3H]NMS concentrations approximately equal to the radioligand equilibrium dissociation constant (Kd) for the different muscarinic receptors subtypes. The [3H]NMS concentration is 0.3 nM for the M1 and M4 assays and 1 nM for the M2, M3, and M5 assays. A range of antagonist concentrations (10−14 to 10−5 M) are tested in duplicate to generate competition curves. Nonspecific binding is determined in the presence of atropine (1 μM). Assay reagents are dissolved in assay binding buffer (phosphate-buffered saline with calcium and magnesium) to a total volume of 200 μL. After a 2 hours or 6 hours incubation period (M1–M4 and M5, respectively) at room temperature in 96-well microtiter plates to ensure that equilibrium is achieved for Aclidinium, 150 μL aliquots of the reaction are transferred to GF/C filter plates pretreated for 1 hour with wash buffer (50 mM Tris, 100 mM NaCl, pH 7.4) containing 0.05% polyethylenimine. Bound and free [3H]NMS are then separated by rapid vacuum filtration followed by four washes with ice-cold wash buffer. Filters are then dried for 30 min before addition of 30 μL of OptiPhase Supermix, and radioactivity is quantified using a MicroBeta Trilux microplate scintillation counter. 

Cell Assay: Human bronchial fibroblast proliferation is measured as previously outlined by colorimetric immunoassay based on BrdU incorporation during DNA synthesis using a cell proliferation enzyme-linked immunosorbent assay BrdU kit according to the manufacturer’s protocol. Cells are seeded at a density of 3x103 cells/well on 96-well plates and incubated for 24 hours. Cells are then exposed to different experimental conditions. The 490 nm absorbance is quantified using a microplate spectrophotometer. Proliferation data refers to the absorbance values of BrdU-labeled cellular DNA content per well. Stimulation is expressed as x-fold proliferation over basal growth of the untreated control set as unity.