Anti-Inflammatory and Immunomodulatory Potential of the Novel PDE4 Inhibitor Roflumilast in Vitro
Abstract
From a series of benzamide derivatives, roflumilast (3-cyclo-propylmethoxy-4-difluoromethoxy-N-[3,5-di-chloropyrid-4-yl]-benzamide) was identified as a potent and selective PDE4 inhibitor. It inhibits PDE4 activity from human neutrophils with an IC50 of 0.8 nM without affecting PDE1 (bovine brain), PDE2 (rat heart), and PDE3 and PDE5 (human platelets) even at 10,000-fold higher concentrations. Roflumilast is almost equipotent to its major metabolite formed in vivo (roflumilast N-oxide) and piclamilast (RP 73401), however, more than 100-fold more potent than rolipram and Ariflo (cilomilast; SB 207499). The anti-inflammatory and immunomodulatory potential of roflumilast and the reference compounds was investigated in various human leukocytes using cell-specific responses: neutrophils [N-formyl-methyl-leucyl-phenylalanine (fMLP)-induced formation of LTB4 and reactive oxygen species (ROS)], eosinophils (fMLP- and C5a-induced ROS formation), monocytes, monocyte-derived macrophages, and dendritic cells (lipopolysaccharide-induced tumor necrosis factor-α synthesis), and CD4+ T cells (anti-CD3/anti-CD28 monoclonal antibody-stimulated proliferation, IL-2, IL-4, IL-5, and interferon-γ release). Independent of the cell type and the response investigated, the corresponding IC values (for half-maximum inhibition) of roflumilast were within a narrow range (2–21 nM), very similar to roflumilast N-oxide (3–40 nM) and piclamilast (2–13 nM). In contrast, cilomilast (40–3000 nM) and rolipram (10–600 nM) showed greater differences with the highest potency for neutrophils. Compared with neutrophils and eosinophils, representing the terminal inflammatory effector cells, the relative potency of roflumilast and its N-oxide for monocytes, CD4+ T cells, and dendritic cells is substantially higher compared with cilomilast and rolipram, probably reflecting an improved immunomodulatory potential. The efficacy of roflumilast in vitro and in vivo (see accompanying article in this issue) suggests that roflumilast will be useful in the treatment of chronic inflammatory disorders such as asthma and chronic obstructive pulmonary disease.
Footnotes
-
Send reprint requests to: Dr. Armin Hatzelmann, Byk Gulden, Department of Biochemistry, P.O. Box 100301, 78403 Konstanz, Germany. E-mail: armin.hatzelmann{at}byk.de
-
This work is dedicated to the inventor of roflumilast, Dr. Hermann Amschler, who deceased in 1999 to our deepest regret.
- Abbreviations:
- PDE
- phosphodiesterase
- COPD
- chronic obstructive pulmonary disease
- BSA
- bovine serum albumin
- PBS
- phosphate-buffered saline
- fMLP
- N-formyl-methionyl-leucyl-phenylalanine
- LPS
- lipopolysaccharide
- PGE2
- prostaglandin E2
- FBS
- fetal bovine serum
- IL
- interleukin
- GM-CSF
- granulocyte macrophage-colony stimulating factor
- DMSO
- dimethyl sulfoxide
- CL
- chemiluminescence
- AUC
- area under the curve
- MACS
- magnetic cell separation
- TNFα
- tumor necrosis factor-α
- mAb
- monoclonal antibody
- IFNγ
- interferon-γ
- LTB4
- leukotriene B4
- ROS
- reactive oxygen species
- HARBS
- high-affinity rolipram binding site
- LARBS
- low-affinity rolipram binding site
-
- Received September 15, 2000.
- Accepted December 4, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
