SKF-94120

As part of our efforts to understand the regulation of intracellular cAMP and to generate new targets for pharmacological intervention, we have cloned and characterized the first isozyme in a new family of cyclic nucleotide phosphodiesterases, PDE8A. PDE8A is most similar to PDE4 (38.5% amino acid identity in the catalytic domain), but is clearly not a member of any of the seven known PDE families. We report the cloning of human cDNA encoding the C-terminal 713 amino acids of the protein, including a 283 amino acid region located near the C-terminus that is homologous to the approximately 270 amino acid catalytic domain of other PDEs. In addition, we found cDNA sequences consistent with alternative 5' mRNA splicing analogous to that seen in other PDE genes. PDE8A is expressed in a wide variety of tissues as a approximately 4.5 kb mRNA, with highest levels in testis, ovary, small intestine, and colon. The C-terminal 545 amino acids of PDE8A (the region shared among all splice variants) were expressed in baculovirus. Kinetic analysis of the baculovirus expressed enzyme shows it to be a very high affinity cAMP specific PDE with a Km of 55 nM for cAMP and 124 microM for cGMP. The Vmax (150 pmol/min/microgram recombinant enzyme) is about 10 times slower than that of PDE4. The cAMP hydrolytic activity of PDE8A is not modulated by cGMP at concentrations up to 100 microM. The enzyme requires the presence of at least 1 mM Mn2+ or Mg2+ for maximal activity in vitro, while 100 mM Ca2+ restores only about 20% activity. PDE8A is insensitive (up to 100 microM) to a variety of PDE inhibitors including rolipram, zaprinast, vinpocetine, SKF-94120, and IBMX, but is inhibited (IC50 = 9 microM) by the PDE inhibitor dipyridimole. To give PDE8A a descriptive name that distinguishes it from the other two known high affinity cAMP-specific phosphodiesterases (PDE4 and PDE7), we denote PDE8A as the high affinity cAMP-specific and IBMX-insensitive PDE.

There is currently interest in the use of inhibitors of cyclic nucleotide phosphodiesterases (PDE) as potential anti-asthma agents. In this study we examined the effects of SCA40 (6-bromo-8-methylaminoimidazol-[1,2-a] pyrazine-2-carbonitrile), a preferential inhibitor of PDE 3 also endowed with PDE 4 and 5 inhibitory activities, on isolated bronchus and eosinophil functions and in an animal model of asthma. SCA40 (1 nM-0.1 mM) produced concentration-dependent inhibition of spontaneous and stimulated tone of human isolated bronchus and reached a maximal relaxation similar to that of theophylline (3 mM). The potency (-log EC50 values) of SCA40 against spontaneous tone (6.52 +/- 0.10) was greater than against tone raised by equieffective concentrations (approximately 70%) of histamine (5.76 +/- 0.06), leukotriene C4 (5.44 +/- 0.11), and acetylcholine (4.98 +/- 0.09). In the presence of cytochalasin B, the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 0.5 microM) induced leukotriene C4 production in human eosinophils isolated in discontinuous metrizamide gradients. The production of leukotriene C4 was inhibited by SCA40 in a concentration-related fashion (-log IC50 = 6.04 +/- 0.20; n = 6). Rolipram, a selective PDE 4 inhibitor, was also effective (-log IC50 = 7.29 +/- 0.32) but the selective PDE 3 inhibitor SKF94120 was scarcely effective (< 10% inhibition for 10 microM). In ovalbumin sensitized guinea-pigs, SCA40 (1 mg kg(-1), i.p.) given 30 min before antigen challenge significantly inhibited the acute bronchoconstriction produced by aerosol antigen (5 mg ml(-1), 30 s) (antigen response was 185 +/- 13 and 91 +/- 21 cmH2O l(-1) s(-1) in control and SCA40-treated animals, respectively, P < 0.05). Pretreatment with SCA40 (1 mg kg(-1), i.p., 30 min pre- and 3 h post-antigen exposure) prevented airway hyperreactivity to histamine which developed 24 h after exposure of conscious guinea-pigs to aerosol antigen. Eosinophil lung accumulation that accompanied airway hyperreactivity was also inhibited by SCA40 (from 6.15 +/- 0.86 in control to 1.27 +/- 0.27 in treated animals; expressed as eosinophils x 10(6); P < 0.05). SCA40 (1 mg kg(-1), i.p.) also inhibited the microvascular leakage produced after inhaled antigen (5 mg ml(-1), 30 s) at all airway levels. The haemodynamic effects of SCA40 (1 mg kg(-1), i.p.) consisted of a rapid decrease (peak at 5 min) in mean arterial blood pressure (-39.4 +/- 2.4%) and tracheal mucosal blood flow (-13.5 +/- 2.0%) that slowly recovered with time. These data support previous work showing that PDE inhibition results in antispasmogenic and anti-inflammatory effects. SCA40 was effective in vitro and in vivo and these effects are probably related to its activity as a mixed PDE inhibitor.