Indacaterol maleate

Ciclesonide (CIC) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Indacaterol maleate (IND) was provided from Novartis Pharma AG (Basel, Switzerland). Poly I:C and dimethyl sulfoxide (DMSO) were purchased from SIGMA-ALDRICH (St. Louis, MO, USA). Anti-mouse CD16/CD32 mAb was purchased from BD Biosciences (San Diego, CA, USA). FITC-conjugated mouse anti-keratin 5/8 monoclonal Ab (mAb) was purchased from Progen (Heidelberg, Germany). Biotinylated anti-human PD-L1 mAb and anti-mouse PD-L1 mAb were purchased from eBioscience (San Diego, CA, USA). Streptavidin-phycoerythrin (SAv-PE) conjugate was purchased from BD Biosciences (San Jose, CA, USA).

All the chemicals used were of analytical reagent grade, and the solvents were of HPLC grade. Indacaterol maleate and glycopyrronium bromide reference substances were kindly provided by Novartis (Basel, Switzerland). Tenoxicam (TNX) as internal standard and maleic acid were obtained from Sigma Chemicals. Inhaler capsules containing 110 µg of IND and 50 µg of GLY/capsule (Ultibro^® Breezhaler^®), 150 µg of IND/capsule (Onbrez Breezhaler^®) and 50 µg of GLY/capsule (Seebri^® Breezhaler^®) were obtained from commercial sources. Acetonitrile and methanol were purchased from Sigma-Aldrich (Germany). Orthophosphoric acid (85% w/v) was obtained from Riedel-deHaën (Sleeze, Germany). Hydrochloric acid (32% w/v), hydrogen peroxide (10% w/v), sodium hydroxide and sodium dihydrogen phosphate were obtained from Adwic Co. (Cairo, Egypt). High purity distilled water was used in the study.

Formoterol fumarate dehydrate and salbutamol hydrochloride were purchased from Sigma-Aldrich (Pty) Ltd. (Johannesburg, South Africa), while indacaterol maleate was provided by Novartis International AG, Basel, Switzerland. All 3 agents were dissolved to a stock concentration of 10 mM in dimethylsulfoxide (DMSO) and used at final concentrations of 1, 10, and 100 nM in most of the assays described below and at 1–1000 nM in others. The local concentration of formoterol following inhalation of 24 μg of this agent has been estimated to be approximately 11 nM [16 (link)]. The final concentration of DMSO in each assay was 0.1% and appropriate solvent controls were included with each experimental system. Unless indicated, all other chemicals and reagents were purchased from Sigma-Aldrich.
Neutrophils were activated with the chemoattractant, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, 1 μM final) either alone or in combination with cytochalasin B (CB, 1 μM final) in assays of oxygen consumption and degranulation. An additional chemoattractant, platelet-activating factor (PAF, 200 nM final) was used in a more limited series of experiments.