Comparison of inhibition of ovalbumin-induced bronchoconstriction in guinea pigs and in vitro inhibition of tumor necrosis factor-alpha formation with phosphodiesterase 4 (PDE4) selective inhibitors.
Phosphodiesterase 4 (PDE4) inhibitors elevate cyclic adenosine 5'-monophosphate (cAMP), and this elevation has been shown to inhibit inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). Using TNF-alpha as a biomarker, we have developed transcription-based assays to examine inhibition of PDE4 activity in human and guinea pig whole blood. In vitro inhibition by PDE4 inhibitors was measured using quantitative PCR (qPCR) analysis of TNF-alpha mRNA levels in whole blood stimulated with lipopolysaccharide (LPS). The kinetics of human TNF-alpha mRNA production were analyzed and shown to be highest 4 hr following LPS stimulation. The guinea pig displayed kinetics of TNF-alpha transcription similar to those of the human. Analysis of inhibition of human TNF-alpha protein production was performed by immunoassay and shown to correlate with inhibition of transcription for three of the four compounds tested. Roflumilast was found to be 9-fold more potent for TNF-alpha inhibition in the qPCR assay than in the protein assay. The potencies of L-826,141 and roflumilast were determined in human and guinea pig whole blood by qPCR, with IC(50) values of 270 and 20 nM, respectively, in humans and 100 and 10 nM, respectively, in guinea pigs. These results show that the potency of PDE4 inhibitors can be monitored in whole blood using a transcription-based assay, and that this type of assay can be adapted to various species provided the TNF-alpha nucleotide sequence is known. The in vitro whole blood IC(50) for TNF-alpha inhibition was compared to inhibition in the ovalbumin-challenged guinea pig model of bronchoconstriction. Obtaining plasma levels at the IC(50) determined in vitro for L-826,141 and roflumilast provides significant inhibition of bronchoconstriction. This suggests that TNF-alpha can be used as a whole blood biomarker in the guinea pig for PDE4 inhibition in this inflammatory model.
Muise
Eric S
ES
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, P.O. Box 1005, Pointe-Claire-Dorval, Quebec, Canada H9R 4P8.
Chute
Ian C
IC
Claveau
David
D
Masson
Paul
P
Boulet
Louise
L
Tkalec
Lydia
L
Pon
Douglas J
DJ
Girard
Yves
Y
Frenette
Richard
R
Mancini
Joseph A
JA
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D016084:Bronchoconstriction / Q000187:drug effects* / Q000502:physiology; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D004347:Drug Interactions; D005260:Female; D006168:Guinea Pigs; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D010047:Ovalbumin / Q000494:pharmacology*; D010726:Phosphodiesterase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D011725:Pyridines / Q000737:chemistry / Q000494:pharmacology*; D012333:RNA, Messenger / Q000097:blood / Q000187:drug effects; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
C459562:L-826,141; D008070:Lipopolysaccharides; D010726:Phosphodiesterase Inhibitors; D011725:Pyridines; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D010047:Ovalbumin; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4