JPET #161919
Pharmacological Blockade of the DP2 Receptor Inhibits Cigarette Smoke-Induced Inflammation, Mucus Cell Metaplasia and Epithelial Hyperplasia in the Mouse Lung
Karin J. Stebbins, Alex R. Broadhead, Christopher S. Baccei, Jill M. Scott, Yen P. Truong, Heather Coate, Nicholas S. Stock, Angelina M. Santini, Patrick Fagan, Patricia Prodanovich, Gretchen Bain, Brian A. Stearns, Christopher D. King, John H. Hutchinson, Peppi Prasit, Jilly F. Evans and Daniel S. Lorrain.
Supplemental Methods
COX-1 human whole blood assay
Human blood was obtained by venipuncture from consenting adult volunteers and collected into heparinized tubes. Aliquots of fresh blood (150 µL) were incubated for 15 minutes at 37°C with 1 µL of DP2 antagonist diluted in DMSO or with DMSO alone as a vehicle control. Calcium ionophore A23187 (25 µM final concentration, Sigma-Aldrich, St. Louis, MO) was added for 30 minutes at 37°C. Incubation mixtures were centrifuged (524g, 10 minutes, 4°C) and aliquots of the plasma were diluted 1:100 with assay buffer and analyzed for TXB2 (a stable metabolite of TXA2) by a competitive enzyme immunoassay according to the manufacturers instructions (Assay Designs, Ann Harbor, MI).
COX-2 human whole blood assay
Aliquots of fresh blood (150 µL) were incubated for 15 minutes at 37°C with 1 µL of DP2 antagonist diluted in DMSO or with DMSO alone as a vehicle control. Lipopolysaccharide (LPS, E. Coli, serotype 0111:B4, Sigma-Aldrich, St. Louis, MO) (100 µg/mL final concentration) was added and incubated for 24 hours at 37°C. Incubation mixtures were centrifuged (524g, 10 minutes, 4°C) and aliquots of the plasma were diluted 1:100 with assay buffer and analyzed for PGE2 by a competitive enzyme immunoassay according to the manufacturers instructions (Assay Designs, Ann Harbor, MI).
Cerep counterscreens
A 25 M stock solution of AM156 was prepared in DMSO and submitted to Cerep (Seattle, WA) for evaluation of membrane receptor binding of 10 M AM156 to a panel of 55 GPCRs, ion channels and transporters included in the ExpresSProfile. A 25 mM stock solution of AM156 was prepared in DMSO and submitted to Cerep (Seattle, WA) for evaluation of inhibition of enzyme activity of elastase, cathepsin G, human neutrophil elastase, kallikrein and tryptase at 25 M.
Supplemental Tables
Supplemental Table 1. Percent inhibition of radioligand binding by 10 M AM156 in Cerep ExpresSProfile counterscreen
Assay / % Inhibition of ControlSpecific Binding / Assay / % Inhibition of Control
Specific Binding
A1 (h) / 33 / NT1 (h) (NTS1) / 2
A2A (h) / 7 / 2 (h) (DOP) / 3
A3 (h) / -4 / (KOP) / 2
1 (non-selective) / 6 / (h) (MOP) (agonist site) / -4
2 (non-selective) / -2 / ORL1 (h) (NOP) / -4
1 (h) / 10 / EP1 (h) / 2
2 (h) / -1 / EP2 (h) / -1
AT1 (h) / 12 / EP4 (h) / 3
BZD (central) / -3 / FP (h) / 29
B2 (h) / -3 / TP (h) (TXA2/PGH2) / 2
CB1 (h) / -4 / IP (PGI2) (h) / -4
CCKA (h) (CCK1) / -46 / 5-HT1A (h) / 5
D1 (h) / -3 / 5-HT1B / 5
D2S (h) / 0 / 5-HT2A (h) / 17
ETA (h) / -13 / 5-HT2B (h) (agonist site) / 19
GABA (non-selective) / -3 / 5-HT3 (h) / -1
GAL2 (h) / 3 / 5-HT5A (h) / 3
CXCR2 (IL-8B) (h) / 1 / 5-HT6 (h) / -8
CCR1 (h) / -4 / 5-HT7 (h) / 2
H1 (h) / 1 / sst (non-selective) / 2
H2 (h) / 5 / VIP1 (h) (VPAC1) / -1
MC4 (h) / -3 / V1a (h) / -3
MT1 (ML1A) (h) / 7 / Ca2+ channel (L, verapamil site) (phenylalkamine) / 1
M1 (h) / 6 / KV channel / 4
M2 (h) / 3 / SKCa channel / -12
M3 (h) / 3 / Na+ channel (site 2) / -15
NK2 (h) / -8 / Cl- channel (GABA-gated) / 2
NK3 (h) / 6 / Norepinephrine transporter (h) / 4
Y1 (h) / 8 / Dopamine transporter (h) / 4
Y2 (h) / -18 / 5-HT transporter (h) / 4
Supplemental Table 2. AM156 counterscreen for COX-1, COX-2 and Cerep enzyme inhibition assays
Assay / % of Control Values / IC50COX-1a / n.d. / > 100 M
COX-2 a / n.d. / 300 M
Elastaseb (h) / 96.9 / n.d.
Cathepsin G b (h) / 95.5 / n.d.
HNE b (h) (human neutrophil elastase) / 107.5 / n.d.
Kallikrein b (h) / 100.3 / n.d.
Tryptase b (h) / 99.4 / n.d.
a Assays performed at Amira Pharmaceuticals. b Assays performed at Cerep. n.d.=not determined