Martin Perry

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

1 The novel tri‐aryl ethane CDP840, is a potent and selective inhibitor of cyclic AMP phosphodiesterase type 4 (PDE 4) extracted from tissues or recombinant PDE 4 isoforms expressed in yeast (IC50s: 4–45 nM). CDP840 is stereo‐selective since its S enantiomer (CT 1731) is 10–50 times less active against all forms of PDE 4 tested while both enantiomers are inactive (IC50s: > 100 μm) against PDE types 1, 2, 3 and 5. 2 Oral administration of CDP840 caused a dose‐dependent reduction of interleukin‐5 (IL‐5)‐induced pleural eosinophilia in rats (ED50 = 0.03 mg kg−1). The eosinophils in pleural exudates from CDP840‐treated animals contained higher levels of eosinophil peroxidase (EPO) than cells from control animals, suggesting a stabilizing effect on eosinophil degranulation. CDP840 was approximately equi‐active with the steroid dexamethasone in this model and was 10–100 times more potent than the known PDE 4‐selective inhibitors rolipram and RP73401. The activity of CDP840 was not influenced by adrenalectomy, β‐sympathomimetics or β‐sympatholytics. 3 Antigen‐induced pulmonary eosinophilia in sensitized guinea‐pigs was reduced dose‐dependently by CDP840 (0.01‐1 mg kg−1, i.p.) and intracellular EPO levels were significantly higher. CDP840 was more potent in these activities than CT1731 or rolipram and comparable in potency to RP73401. 4 Rolipram or CDP840 were less active than dexamethasone in preventing neutrophil accumulation, or exudate formation in carrageenan‐induced pleurisy in rats and thus do not exhibit general antiinflammatory activity. 5 In sensitized guinea‐pigs, aerosols of the antigen ovalbumin caused a dose‐dependent bronchoconstriction demonstrated by an increase in pulmonary inflation pressure. Administration of CDP840 (0.001‐1.0 mg kg−1, i.p.), 1 h before antigen challenge, resulted in dose‐dependent reduction in response to antigen. This activity was not due to bronchodilatation since higher doses of CDP840 (3 mg kg−1) did not significantly change the bronchoconstrictor response to histamine. Rolipram was approximately 10 times less active than CDP840 in preventing antigen‐induced bronchoconstriction. 6 These results confirm the observations that selective PDE 4 inhibitors reduce antigen‐induced bronchoconstriction and pulmonary eosinophilic inflammation. CDP840 is more potent than rolipram in inhibiting native or recombinant PDE 4. Unlike the recently described potent PDE 4 inhibitor RP73401, CDP840 is more active than rolipram in the rat IL‐5 model following oral administration. The novel series of tri‐aryl ethanes, of which CDP840 is the lead compound, could be the basis of an orally active prophylactic treatment for human asthma.

PDE 4 inhibitors: the use of molecular cloning in the design and development of novel drugs

Phosphodiesterase 4 (PDE 4) enzymes are the principal phosphodiesterases responsible for the hydrolysis of cAMP in pro-inflammatory leukocytes. The functional consequences of elevating cAMP in these cells suggests that inhibition of PDE 4 offers a novel approach to asthma therapy. However, clinical development of early inhibitors has been limited by the side-effect of nausea. In this review, we detail how the molecular biology of the PDE 4 gene family has been integrated with biochemical, cellular and pharmacological studies. This approach has led to the discovery and development of CDP840, a prototype inhibitor for which efficacy has been demonstrated in a clinical model of asthma in the absence of side-effects.

Development of a recombinant cell-based system for the characterisation of phosphodiesterase 4 isoforms and evaluation of inhibitors

We described the development of a recombinant cell-based system for the characterisation of phosphodiesterase (PDE) 4 isoforms and the evaluation of inhibitors. The Chinese hamster ovary (CHO) cell, which was found to have a low endogenous PDE4 background and no beta-adrenergic receptors (beta-AR), was transiently transfected with beta-AR and various PDE4 isoforms which were expressed as functionally coupled molecules. From correlations of elevation of adenosine 3,5-cyclic monophosphate in situ and the inhibition of catalytic activity in vitro with the various PDE4 isoforms, it was apparent that PDE4A4, 4B2, 4C2, 4D2, and 4D3 all adopted a high-affinity binding conformation (i.e. expressed the high-affinity rolipram binding site) in the CHO cell, whereas PDE4A330 was expressed in a low-affinity conformation in situ. This gives the opportunity of using this system to screen and optimise inhibitors against a low-affinity conformation of PDE4 in situ and use a high-affinity conformation of PDE4 as a counterscreen, as inhibitor activity against this conformer has been linked with undesirable side effects. This system could also be utilised to screen inhibitors against various PDE4 isoforms in isolation against a low endogenous PDE background in situ for isoform-selective inhibitors.

Molecular Cloning and Expression of a Human Phosphodiesterase 4C

A cDNA coding for a human phosphodiesterase 4C (PDE4C2) was isolated from the mRNA prepared from the glioblastoma cell line, U87. The cDNA contained an ORF of 1818 bp corresponding to a 605 amino acid polypeptide. The sequence differed at the 5 end from the human PDE4C previously reported (Engels, P. et al, 1995 FEBs Letters 358, 305-310) indicating that it represents a novel splice variant of the human PDE4C gene. Evidence was also obtained for a third 5 splice variant. The PDE4C2 cDNA was transfected into both COS 1 cells and yeast cells, and shown to direct the expression of an 80 kD polypeptide by Western blotting using a PDE4C specific antiserum. The activity of cell lysates was typical of PDE4 being specific for cAMP and inhibitable by the selective inhibitor, rolipram. However, the Km for cAMP of the enzyme produced in COS cells was 0.6 microM compared to 2.6 microM for the yeast 4C activity. In addition the COS cell PDE4 activity was much more sensitive to R rolipram than the yeast PDE4 enzyme (IC50 of 23 nM compared to 1648 nM). This difference in rolipram sensitivity was associated with the detection of a high affinity [3H] R rolipram binding site on the COS cell 4C enzyme but not on the yeast expressed enzyme. The results indicate that the enzyme can adopt more than one active conformation, which are distinguished by their interaction with rolipram.

CDP840: a novel inhibitor of PDE-4.

We present the in vitro characterization of a novel phosphodiesterase type 4 inhibitor, CDP840 (R-[+]-4-[2-¿3-cyclopentyloxy-4-methoxyphenyl¿-2-phenylethyl]pyridine), which has shown efficacy in a phase II allergen challenge study in asthmatics without adverse effects. CDP840 potently inhibits PDE-4 isoenzymes (IC50 2-30 nM) without any effect on PDE-1, 2, 3, 5, and 7 (IC50 > 100 microM). It exhibited no significant selectivity in inhibiting human recombinant isoenzymes PDE-4A, B, C or D and was equally active against the isoenzymes lacking UCR1 (PDE-4B2 and PDE-4D2). In contrast to rolipram, CDP840 acted as a simple competitive inhibitor of all PDE-4 isoenzymes. Studies with rolipram indicated a heterogeneity within all the preparations of PDE-4 isoenzymes, indicative of rolipram inhibiting the catalytic activity of PDE-4 with both a low or high affinity. These observations were confirmed by the use of a PDE-4A variant, PDE-4A330-886, which rolipram inhibited with low affinity (IC50 = 1022 nM). CDP840 in contrast inhibited this PDE-4A variant with similar potency (IC50 = 3.9 nM), which was in good agreement with the Kd of 4.8 nM obtained from [3H]-CDP840 binding studies. Both CDP840 and rolipram inhibited the high-affinity binding of [3H]-rolipram binding to PDE-4A, B, C, and D with similar Kd app (7-19 nM and 3-5 nM, respectively). Thus, the activity of CDP840 at the [3H]-rolipram binding site was in agreement with the inhibitors activity at the catalytic site. However, rolipram was approximately 100-fold more potent than CDP840 at inhibiting the binding of [3H]-rolipram to mouse brain in vivo. These data clearly demonstrate that CDP840 is a potent selective inhibitor of all PDE-4 isoenzymes. In contrast to rolipram, CDP840 was well-tolerated in humans. This difference, however, cannot at present be attributed to either isoenzyme selectivity or lack of activity in vitro at the high-affinity rolipram binding site (Sr).

4-pyridin-5-yl-2-(3,4,5-trimethoxyphenylamino)pyrimidines : Potent and selective inhibitors of ZAP 70

Activation of the tyrosine kinase ZAP 70 has been shown to be crucial to the transduction of the T-cell receptor signalling pathway, which leads ultimately to proliferation, cytokine gene expression and T-cell effector functions. A series of 2-phenylaminopyrimidines have been identified as potent and selective inhibitors of ZAP 70.