Suppression of eosinophil function by RP 73401, a potent and selective inhibitor of cyclic AMP‐specific phosphodiesterase: comparison with rolipram

Abstract

1. We have investigated the inhibitory potency of RP 73401, a novel, highly selective and potent inhibitor of cyclic AMP-specific phosphodiesterase (PDE IV), against partially-purified PDE isoenzymes from smooth muscle and the particulate PDE IV from guinea-pig eosinophils. The inhibitory effects of RP 73401 on the generation of superoxide (.O2-), major basic protein (MBP) and eosinophil cationic protein (ECP) from guinea-pig eosinophils have also been studied. 2. RP 73401 potently inhibited partially-purified cyclic AMP-specific phosphodiesterase (PDE IV) from pig aortic smooth muscle (IC50 = 1.2 nM); it was similarly potent against the particulate PDE IV from guinea-pig peritoneal eosinophils (IC50 = 0.7 nM). It displayed at least a 19000 fold selectivity for PDE IV compared to its potencies against other PDE isoenzymes. Rolipram was approximately 2600 fold less potent than RP 73401 against pig aortic smooth muscle PDE IV (IC50 = 3162 nM) and about 250 times less potent against eosinophil PDE IV (IC50 = 186 nM). 3. Solubilization of the eosinophil particulate PDE IV increased the potency of rolipram 10 fold but did not markedly affect the potency of RP 73401. A similar (10 fold) increase in the PDE IV inhibitory potency of rolipram, but not RP 73401, was observed when eosinophil membranes were exposed to vanadate/glutathione complex (V/GSH). 4. Reverse transcription polymerase chain reaction (RT-PCR), using primer pairs designed against specific sequences in four distinct rat PDE IV subtype cDNA clones (PDE IVA-D), showed only mRNA for PDE IVD in guinea-pig eosinophils. PDE IVD was also the predominant subtype expressed in pig aortic smooth muscle cells. 5. RP 73401 (Kiapp = 0.4 nM) was 4 fold more potent than (+/-)-rolipram (Kiapp = 1.7 nM) in displacing[3H]-(+/-)-rolipram from guinea-pig brain membranes.6. In intact eosinophils, RP 73401 potentiated isoprenaline-induced cyclic AMP accumulation(EC50 = 79 nM). RP 73401 also inhibited leukotriene B4-induced generation of *02- (IC50 = 25 nM), and the release of major basic protein (ICo = 115 nM) and eosinophil cationic protein (IC50 = 7 nM). Rolipram was 3-14 times less potent than RP 73401.7. Thus RP 73401 is a very potent and selective PDE IV inhibitor which suppresses eosinophil function suggesting that it may be a useful agent for the treatment of inflammatory diseases such as asthma. The greatly different inhibitory potencies of rolipram against PDE IV from smooth muscle and eosinophils(in contrast to the invariable effects of RP 73401) are unlikely to be attributable to diverse PDE IV subtypes but suggest distinct interactions of the two inhibitors with the enzyme.

The inhibition of antigen‐induced eosinophilia and bronchoconstriction by CDP840, a novel stereo‐selective inhibitor of phosphodiesterase type 4

“…It is known, for example, that rolipram is relatively more active in elevating cyclic AMP and inhibiting superoxide production in eosinophils than it is against the cell-free enzyme (Souness et al, 1991). Furthermore, solubilization of particulate eosinophil PDE 4 or treatment of eosinophil membranes with vanadate/ glutathione results in a ten fold increase in the potency of rolipram, indicating that the conformation of PDE 4 determines its susceptibility to inhibitors (Souness et al, 1995).…”

“…IL-5 released from allergenspecific T-lymphocytes in the lungs of asthmatics is thought to be a local chemotactic mechanism for the recruitment of eosinophils (Corrigan & Kay, 1990). The elevation of intracellular cyclic AMP in eosinophils by selective PDE 4 inhibitors has been associated with suppression of eosinophil function in vitro (Souness et al, 1995) and inhibition of eosinophil recruitment in vivo (Underwood et al, 1993 (Pober et al, 1993).…”

The effects of phosphodiesterase type 4 inhibitors on tumour necrosis factor‐α and leukotriene B4 in a novel human whole blood assay

“…RS14203 (Wilhelm & Fatheree, 1994), RP73401 (Souness et al, 1995), T-440 (Kaminuma et al, 1996), R-and S-rolipram, SB207499 (Christensen et al, 1998), CDP840 (Hughes et al, 1996), CT1731 (Hughes et al, 1996), Trequinsin, L-739,010 (Hamel et al, 1997), MF-tricyclic (selective cyclo-oxygenase-2 (COX-2) inhibitor) (Oshima et al, 1996) Figure 1 shows the time course of TNF-a and PGE 2 production after LPS-stimulation of whole blood. During the incubation period, a sharp rise in TNF-a levels was observed from 2 h (2.70+0.76 ng ml 71 ) to 4 h (15.59+3.61 ng ml 71 ) and peaked at 8 h (22.92+4.77 ng ml 71 ).…”

Inhibitor Binding to Type 4 Phosphodiesterase (PDE4) Assessed Using [3H]Piclamilast and [3H]Rolipram

“…This is particularly evident when one compares the effects of piclamilast (RP 73,401) and rolipram. Piclamilast is about 1000-fold more potent than rolipram for inhibiting guinea pig smooth muscle PDE4, but only about 4-fold more potent for inhibiting methacholine-induced contraction of guinea pig trachealis muscle or enhancing isoproterenol-stimulated cyclic AMP formation in guinea pig eosinophils (Ashton et al, 1994;Souness et al, 1995).…”