Jan-Anders Karlsson

Anti-inflammatory and bronchodilator properties of RP 73401, a novel and selective phosphodiesterase type IV inhibitor.

1 We have investigated the effects of RP 73401, a novel, potent and highly selective cyclic nucleotide phosphodiesterase (PDE) type IV inhibitor, in guinea‐pig and rat models of bronchoconstriction and allergic inflammation. In some models, the effects of RP 73401 have been compared with those of the standard PDE type IV inhibitor, rolipram. 2 RP 73401 (0.4–400 μg kg−1, intratracheally (i.t.) on lactose) inhibited antigen‐induced bronchospasm in previously sensitized conscious guinea‐pigs (ID50: 7 ± 1 μg kg−1) and in anaesthetized rats (ID50: 100 ± 25 μg kg−1). Rolipram inhibited the antigen‐induced bronchospasm in guinea‐pigs with an ID50 of 5 ± 1 μg kg−1. In guinea‐pig bronchoalveolar lavage (BAL) fluid, total inflammatory cell and eosinophil numbers were reduced by RP 73401 (ID50s: 3.9 ± 0.8 μg kg−1 and 3.2 ± 0.7 μg kg−1, respectively). In the rat, inflammatory cell numbers are less affected. Only the highest dose of RP 73401 (400 μg kg−1) significantly inhibited eosinophil influx (41 ± 16% inhibition). 3 RP 73401 (0.02–100 μg kg−1, i.v.) inhibited PAF‐induced bronchial hyperreactivity to bombesin in the anaesthetized guinea‐pig (ID50: 0.09 ± 0.03 μg kg−1) and inhibited (0.4–40 μg kg−1, i.t.) histamine‐induced airway microvascular leakage in the anaesthetized guinea‐pig by approximately 60% at all doses. 4 RP 73401 relaxed guinea‐pig isolated trachea under basal tone (EC50: 9 nm) and when precontracted with histamine (IC50: 2 nm), methacholine (IC50: 29 nm) or leukotriene D4 (LTD4, IC50: 4 nm). 5 RP 73401 (0.4–100 μg kg−1, i.t.) inhibited bronchospasm induced by histamine (ID50: 34 ± 6 μg kg−1), methacholine (ID50: 66 ± 12 μg kg−1) and LTD4 (ID50: < 4 μg kg−1) in the anaesthetized guinea‐pig. Against these same bronchoconstrictors, rolipram (i.t.) had ID50 values of 44 ± 4, 72 ± 18 and < 4 μg kg−1 respectively. RP 73401 (4 and 40 μg kg−1, i.t.) increased the magnitude and duration of bronchodilatation produced by salbutamol in the anaesthetized guinea‐pig. At doses producing significant bronchodilatation, RP 73401 was without effect on heart rate or blood pressure in the anaesthetized guinea‐pig. RP 73401 (0.01‐0.25 mg kg−1, i.v.) did not affect heart rate and produced only a small fall in blood pressure in the anaesthetized rat. 6 These data demonstrate that RP 73401 and rolipram inhibit antigen‐ and mediator‐induced bronchospasm in guinea‐pigs with the same potency. Furthermore, RP 73401 administered directly into the airways, protects against allergic airway inflammation. These results indicate the importance of PDE IV in regulating smooth muscle and inflammatory cell activity. At doses suppressing the inflammatory response in the lung, RP 73401 had little effect in the cardiovascular system. RP 73401 may have a role as a bronchodilator and, more importantly, as a prophylactic anti‐inflammatory agent in the treatment of asthma.

Evidence that cyclic AMP phosphodiesterase inhibitors suppress TNFα generation from human monocytes by interacting with a ‘low‐affinity’ phosphodiesterase 4 conformer

1 We have investigated the inhibitory effects of RP 73401 (piclamilast) and rolipram against human monocyte cyclic AMP‐specific phosphodiesterase (PDE4) in relation to their effects on prostaglandin (PG)E2‐induced cyclic AMP accumulation and lipopolysaccharide (LPS)‐induced TNFα production and TNFα mRNA expression. 2 PDE4 was found to be the predominant PDE isoenzyme in the cytosolic fraction of human monocytes. Cyclic GMP‐inhibited PDE (PDE3) was also detected in the cytosolic and particulate fractions. Reverse transcription polymerase chain reaction (RT‐PCR) of human monocyte poly (A+) mRNA revealed amplified products corresponding to PDE4 subtypes A and B of which the former was most highly expressed. A faint band corresponding in size to PDE4D was also observed. 3 RP 73401 was a potent inhibitor of cytosolic PDE4 (IC50: 1.5 ± 0.6 nM, n = 3). (±)‐Rolipram (IC50: 313 ± 6.7 nM, n = 3) was at least 200 fold less potent than RP 73401. R‐(−)−rolipram was approximately 3 fold more potent than S‐(+)‐rolipram against cytosolic PDE4. 4 RP 73401 (IC50: 9.2 ± 2.1 nM, n = 6) was over 50 fold more potent than (±)‐rolipram (IC50: 503 ± 134 nM, n = 6)) in potentiating PGE2‐induced cyclic AMP accumulation. R‐(−)−rolipram (IC50: 289 ± 121 nM, n = 5) was 4.7 fold more potent than its S‐(+)‐enantiomer (IC50: 1356 ± 314 nM, n = 5). A strong and highly‐significant, linear correlation (r = 0.95, P < 0.01, n = 13) was observed between the inhibitory potencies of a range of structurally distinct PDE4 inhibitors against monocyte PDE4 and their ED50 values in enhancing monocyte cyclic AMP accumulation. A poorer, though still significant, linear correlation (r = 0.67, P < 0.01, n = 13) was observed between the potencies of the same compounds in potentiating PGE2‐induced monocyte cyclic AMP accumulation and their abilities to displace [3H]‐rolipram binding to brain membranes. 5 RP 73401 (IC50: 6.9 ± 3.3 nM, n = 5) was 71 fold more potent than (±)‐rolipram (IC50: 490 ± 260 nM, n = 4) in inhibiting LPS‐induced TNFα release from monocytes. R‐(−)−rolipram (IC50: 397 ± 178 nM, n = 3) was 5.2‐fold more potent than its S‐(+)‐ enantiomer (IC50: 2067 ± 659 nM, n = 3). As with cyclic AMP, accumulation a closer, linear correlation existed between the potency of structurally distinct compounds in suppressing TNFα with PDE4 inhibition (r = 0.93, P < 0.01, n = 13) than with displacement of [3H]‐rolipram binding (r = 0.65, P < 0.01, n = 13). 6 RP 73401 (IC50: 2 nM) was 180 fold more potent than rolipram (IC50: 360 nM) in suppressing LPS (10 ng ml−1)‐induced TNFα mRNA. 7 The results demonstrate that RP 73401 is a very potent inhibitor of TNFα release from human monocytes suggesting that it may have therapeutic potential in the many pathological conditions associated with over‐production of this pro‐inflammatory cytokine. Furthermore, PDE inhibitor actions on functional responses are better correlated with inhibition of PDE4 catalytic activity than displacement of [3H]‐rolipram from its high‐affinity binding site, suggesting that the native PDE4 in human monocytes exists predominantly in a ‘low‐affinity’ state.

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

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 O2− (IC50 = 25 nM), and the release of major basic protein (IC50‐ 115 nM) and eosinophil cationic protein (IC50 = 7nM). 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.

RPR 106541, a novel, airways-selective glucocorticoid : effects against antigen-induced CD4+T lymphocyte accumulation and cytokine gene expression in the Brown Norway rat lung

1 The effects of a novel 17‐thiosteroid, RPR 106541, were investigated in a rat model of allergic airway inflammation. 2 In sensitized Brown Norway rats, challenge with inhaled antigen (ovalbumin) caused an influx of eosinophils and neutrophils into the lung tissue and airway lumen. In the lung tissue there was also an accumulation of CD4+ T lymphocytes and increased expression of mRNA for interleukin‐4 (IL‐4) and IL‐5, but not interferon‐γ (IFN‐γ). These findings are consistent with an eosinophilia orchestrated by activated Th2‐type cells. 3 RPR 106541 (10–300 μg kg−1), administered by intratracheal instillation into the airways 24 h and 1 h before antigen challenge, dose‐dependently inhibited cell influx into the airway lumen. RPR 106541 (100 μg kg−1) caused a significant (P<0.01) (98%) inhibition of eosinophil influx and a significant (P<0.01) (100%) inhibition of neutrophil influx. RPR 106541 was approximately 7 times and 4 times more potent than budesonide and fluticasone propionate, respectively. 4 When tested at a single dose (300 μg kg−1), RPR 106541 and fluticasone each caused a significant (P<0.01) (100%) inhibition of CD4+ T cell accumulation in lung tissue. Budesonide (300 μg kg−1) had no significant effect. RPR 106541 and fluticasone (300 μg kg−1), but not budesonide (300 μg kg−1), significantly (P<0.05) inhibited the expression within lung tissue of mRNA for IL‐4. RPR 106541 (300 μg kg−1) also significantly (P<0.05) inhibited expression of mRNA for IL‐5. 5 The high topical potency of RPR 106541 in this model, which mimics important aspects of airway inflammation in human allergic asthmatics, suggests that this glucocorticoid may be useful in the treatment of bronchial asthma.

Comparison of the effects of selective inhibitors of phosphodiesterase types III and IV in airway smooth muscle with differing β-adrenoceptor subtypes

1 The relaxant properties of the type IV adenosine 3′,5′‐cyclic monophosphate phosphodiesterase (cyclic AMP PDE) inhibitor, rolipram and the β2‐selective and non‐selective β‐adrenoceptor agonists salbutamol and isoprenaline, were compared on the guinea‐pig, bovine, and mouse trachea and porcine bronchus all precontracted with methacholine (EC30). 2 Rolipram and both β‐agonists produced concentration‐dependent reversal of the methacholine‐induced tone in the four airway preparations. 3 Isoprenaline and salbutamol were similar in potency on the guinea‐pig (−log10IC50:8.43, 8.06) and bovine (−log10 IC50:8.52, 8.40) airways. In contrast, salbutamol was much less potent than isoprenaline on the mouse trachea (> 1000 fold) and the porcine bronchus (> 100 000 fold). 4 The potency of rolipram approached that of isoprenaline on the guinea‐pig and bovine trachea (β2‐adrenoceptors predominate). However, rolipram was significantly less active than isoprenaline on the porcine bronchus (1000 fold) and mouse trachea (> 2000 fold) where β2‐adrenoceptors predominate. 5 Siguazodan, the type III cyclic AMP PDE inhibitor, produced concentration‐dependent relaxations of the porcine bronchus and guinea‐pig trachea contracted with methacholine. Siquazodan was 100 fold more active than rolipram in pig tissues indicating the type III isoenzyme may be of greater functional significance in this tissue. In contrast, siguazodan was 15 times less potent that rolipram in guinea‐pig airways suggesting a greater role for the type IV PDE. 6 These findings may reflect a possible relationship between the β2‐adrenoceptor subtype and the functional importance of the type IV PDE isoenzyme. A similar relationship may exist between β1‐adrenoceptors and the PDE type III isoenzyme.

Effects of rolipram and siguazodan on the human isolated bronchus and their interaction with isoprenaline and sodium nitroprusside

1 The effects of the selective inhibitors of cyclic AMP phosphodiesterase type IV (rolipram) and type III (siguazodan) and their interactions with isoprenaline and sodium nitroprusside have been studied in the human isolated bronchus. 2 On bronchi under resting tone rolipram was, in terms of potency (pD2 = 7.77 ± 0.14, n = 8), very similar to isoprenaline (pD2 = 7.31 ± 0.12, n = 12) and salbutamol (pD2 = 7.12 ± 0.17, n = 10) and approximately 10 fold more potent than siguazodan (pD2 = 6.80 ± 0.12, n = 6). In terms of efficacy (Emax, expressed as percentage of maximal effect induced by theophylline 3 mm), both rolipram and siguazodan were less efficient (Emax = 74 ± 6.7%, n = 8 and 66 ± 7.5%, n = 6, respectively) than isoprenaline (Emax = 98 ± 0.4%, n = 12) and salbutamol (Emax = 83 ± 2.4%, n = 10). 3 During precontraction induced by methacholine (3 × 10−7 m) or acetylcholine (10−3 m), concentration‐response curves to rolipram and siguazodan were shifted to the right and maximal effects reduced. Rolipram was more potent than siguazodan and, in terms of efficacy, it was less active. 4 Rolipram 10−8 and 10−7 m but not siguazodan potentiated the effects of isoprenaline as shown by the shift to the left of the concentration‐response curve to isoprenaline. Sodium nitroprusside‐induced relaxation was not modified by either drug. 5 These results show that rolipram is a potent relaxant of the human isolated bronchus, potentiating the effects of β‐adrenoceptor stimulation and suggest that, as previously demonstrated in other species (guinea‐pig, cow) (Tomkinson et al., 1993), there may be a connection between the β2‐adrenoceptor subtype, which predominate in human airway smooth muscle, and the cyclic AMP phosphodiesterase type IV.

Isozyme-selective cyclic nucleotide phosphodiesterase inhibitors: Biochemistry, pharmacology and therapeutic potential in asthma

Asthma is a disease characterized by airways obstruction resulting from acute constriction of the airways smooth muscle and mucosal inflammation with evident oedema [1]. The inflammation seems to correlate with the severity of the disease and has been linked with hyperresponsiveness to bronchoconstrictor agents [2]. Amongst the prominent features of this bronchial inflammation is an accumulation of inflammatory cell types, particularly eosinophils into the airway tissues and lumen [3, 4]. The numbers of mast cells and sometimes lymphocytes also are increased and, through the release of cytokines, may orchestrate the inflammatory response [5]. A significant proportion of the inflammatory cells may be in their activated, de-granulated state and high levels of cytotoxic proteins such as eosinophil cationic protein (ECP), major basic protein (MBP) and mast cell tryptase can be detected together with albumin, other plasma proteins and a range of putative asthma mediators. The ongoing inflammatory process is fuelled by these cytotoxic proteins and mediators and by extravasated pro-inflammatory proteins found in the inflammatory exudate as a result of leakage from the tracheobronchial microcirculation [6].

Effects of RP 73401, a novel, potent and selective phosphodiesterase type 4 inhibitor, on contractility of human, isolated bronchial muscle

1 The aim of this study was to investigate the smooth muscle relaxant effects of the novel, selective phosphodiesterase (PDE) type 4 inhibitor, RP 73401 in comparison with the classical PDE 4 inhibitor, rolipram, the non‐selective PDE inhibitor, theophylline and the β‐adrenoceptor agonist, isoprenaline on the human, isolated bronchus. 2 At resting tone, the rank order of potency (pD2) for the relaxants was RP 73401 ≥ rolipram ≥ isoprenaline > > theophylline. In terms of maximum relaxation produced (Emax) the PDE 4‐selective inhibitors were similar, but the maximal effects (70–75% of theophylline, 3 mM) were lower than that observed with isoprenaline (98% of theophylline, 3 mM) or theophylline itself (100%). 3 On the human isolated bronchus pre‐contracted with acetylcholine (ACh, 0.1 or 1.0 mM), the rank order of potency remained the same. The maximal responses to RP 73401 and rolipram were however markedly reduced (Emax 39.9–46.6%) compared with isoprenaline (Emax 79–85%). 4 In tissues pre‐contracted with ACh (0.1 mM), RP 73401 and rolipram (10−9‐10−7 m) significantly and concentration‐dependently increased tissue sensitivity to isoprenaline. RP 73401 and rolipram were similar in potency. Both selective PDE 4 inhibitors also significantly increased the maximal relaxant effects of isoprenaline. These effects were not observed with the PDE 3 inhibitor, siguazodan. 5 In terms of retention by tissues (an index of duration of action), the onset of action of RP 73401 (2.11 ± 0.53 min) and rolipram (1.70 ± 0.45 min) was significantly slower than that of isoprenaline (0.33 ± 0.06 min) or theophylline (1.17 ± 0.25 min). The retention of RP 73401 (89.0 ± 21.9 min) on the human isolated bronchial tissues after washing was however dramatically longer than that of rolipram (18.3 ± 4.5 min), theophylline (3.43 ± 0.58 min) or isoprenaline (2.81 ± 0.31 min). 6 These data indicate that RP 73401 is a potent and long‐acting relaxant of human bronchial muscle in vitro. RP 73401 is more potent than the classical PDE 4‐selective inhibitor rolipram and the non‐selective PDE inhibitor theophylline and is retained in bronchial tissue for a much longer period of time.

Potassium channel openers: Airway pharmacology and clinical possibilities in asthma

Asthma is a common disease worldwide, generally affecting 5–10% of the population, and its incidence is, unfortunately, still on the increase. This is despite intensive research into its pathophysiology and the development of several new therapeutic agents. The clinical symptoms of asthma are very obvious and distressing yet we still have only a rudimentary picture of the pathophysiological mechanisms involved. Airway inflammation and hyperreactivity are characteristic features and, since they seem to correlate with the severity of the disease, asthma is now widely regarded as an inflammatory disease [1–3]. Consequently, research into the physiology and pharmacology of airway smooth muscle and the role of bronchodilator drugs has attracted comparatively less interest in recent years. However, the importance of controlling airway smooth muscle contractility to improve airflow and so provide immediate, symptomatic relief for the asthmatic patient is still vital and indeed the bronchodilators remain the first line therapy [3].

IFN-Gamma Production from Human Th1 Cells Is Controlled by Raf Kinase

Raf kinase is an important intracellular mediator in T cell signalling and may be crucial for the proliferation of this inflammatory cell. In order to elucidate its effect on cytokine production by human T cells in response to T cell receptor activation, experiments were carried out on human T cell clones using antisense (AS) oligodeoxynucleotides (ODN) to inhibit the expression of Raf kinase. AS ODN to Raf were shown to have a significant effect on a human Th1-like T cell clone, inhibiting antiCD3-induced IFN-gamma secretion by 76%, whereas no inhibitory effect was observed on IL-5 or IL-4 production by a Th2-like clone. IL-2 secretion from both clones was also not affected by the Raf AS ODN. In all cases, a reduction in Raf kinase within the cell was demonstrated by Western blot. Our results clearly demonstrate the importance of Raf kinase in the production of IFN-gamma from Th1 cells, but also show the lack of effect of this intracellular mediator on cytokine (IL-5, IL-4) release from Th2 cells.