David Raeburn

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.

Time-course of antigen-induced airway inflammation in the guinea-pig and its relationship to airway hyperresponsiveness

The causative relationship between airway inflammation and hyperreactivity is unclear, since inflammatory changes have been examined at one or, at most, a few time-points after antigen challenge in both human asthma and animal models. We have made a detailed investigation of inflammatory and functional changes in the airways up to 8 days after antigen challenge in guinea-pigs. In particular, we examined the hypothesis that eosinophil-derived mediators contribute to tissue damage and the development of airway hyperresponsiveness. Following antigen challenge, the influx of inflammatory cells and mediator release in airway tissue and bronchoalveolar lavage fluid were correlated temporally with histopathological changes in airway tissue and airway responsiveness. Eosinophil influx was demonstrable at 4 h. Eosinophilia peaked after 24 h and persisted for at least 8 days. Parallel increases in the concentrations of major basic protein and eosinophil cationic protein in bronchoalveolar lavage fluid indicated that the eosinophils were activated. Eosinophilia was accompanied by subepithelial oedema and epithelial damage co-localized with major basic protein immunoreactivity. A transient neutrophilia (< 48 h duration) and an increase in neutrophil elastase in bronchoalveolar lavage fluid peaked at 14 h. The proportion of airway macrophages with an activated morphology increased at 8 h and remained markedly elevated until 72 h. Airways were hyperresponsive to histamine at 4 h and for at least 8 days. The antigen-induced airway inflammation resemble in time-course and histopathology that seen in antigen-challenged asthmatics, and indicate that the eosinophil and its cytotoxic proteins may be major mediators of airway mucosal damage and airway hyperresponsiveness.

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.

Techniques for drug delivery to the airways, and the assessment of lung function in animal models

A common approach to understanding the mechanisms underlying clinical asthma and in new drug development is to mimic the disease in animal models. When developing animal models of pulmonary diseases, such as asthma, the experimentally induced disease may be characterized in terms of pathophysiological changes induced (e.g., inflammation, smooth muscle contraction) or by the indices of lung function that are effected by such changes. Similarly, the effects of drugs can be assessed in terms of the reversal of disease- or mediator-induced changes in lung function. Small animals, such as the guinea pig and rat, are commonly used for the assessment of lung function in models of pulmonary diseases, such as asthma, and to evaluate the effects of drugs. A variety of techniques, differing in their level of sophistication, has been developed to measure parameters of lung function in small laboratory animals. Simple techniques involve the visual assessment of the response of a conscious animal to bronchoconstriction induced by an inhaled spasmogen or antigen. This technique is rapid but gives results that are difficult to interpret in physiological terms. Bronchospasm can be better assessed in anesthetized, mechanically ventilated animals by recording bronchial tone as changes in either 1) ventilation circuit pressure or 2) air overflow as the lungs are inflated. These techniques are widely used but because they require surgical intervention they are not suited to long-term or repeat studies. In addition, they give only a limited indication of the physiological changes that affect airway caliber. To improve the models available, researchers have subsequently developed techniques that use the same physiological principles as some of the tests applied to the assessment of lung function in humans. These techniques allow the measurement of parameters of respiratory mechanics, such as lung compliance and airway resistance, that determine the relationship between pulmonary pressure changes and air flow into and out of the lungs. Continued development has resulted in models that use nonsurgical plethysmographic techniques. These allow the long-term or repeated measurement of lung function in conscious animals under minimal restraint. In the treatment of asthma, inhalation is the preferred route of administration of a drug as it allows rapid drug delivery to the site of action. Systemic effects are reduced, and the therapeutic dose is minimized. Drugs are generally inhaled as either nebulized liquids or dry-powder formulations. Because drug inhalation requires patient cooperation, techniques have been modified to allow drug delivery to the airways of experimental animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Possible role of cyclic AMP phosphodiesterases in the actions of ibudilast on eosinophil thromboxane generation and airways smooth muscle tone

1 The possible role of cyclic AMP phosphodiesterase (PDE) in the inhibitory actions of ibudilast on tracheal smooth muscle contractility and eosinophil thromboxane generation was investigated. 2 Ibudilast was a non‐selective inhibitor of partially purified cyclic nucleotide PDE isoenzymes from pig aorta and bovine tracheal smooth muscle, exhibiting only moderate potency against bovine tracheal PDE IV (IC50 = 12 ± 4 μm, n = 3). Similar or slightly lower potencies were displayed against PDEs I, II, III and V. In contrast, rolipram exhibited selectivity for PDE IV (3 ± 0.5 μm, n = 3). 3 Ibudilast (IC50 = 0.87 ± 0.37 μm, n = 3), like rolipram (IC50 = 0.20 ± 0.04 μm, n = 3), was a more potent inhibitor of membrane‐bound PDE IV from guinea‐pig eosinophils than of partially purified PDE IV from bovine tracheal smooth muscle. The potency of ibudilast increased when the eosinophil enzyme was solubilised with deoxycholate and NaCl (IC50 = 0.11 ± 0.05 μm, n = 3) or exposed to vanadate/glutathione complex (V/GSH) (IC50 = 0.11 ± 0.02 μm, n = 3). The potency of rolipram was also increased by solubilization (IC50 = 0.012 ± 0.003, n = 3) or V/GSH (IC50 = 0.012 ± 0.003, n = 3). 4 In intact eosinophils, ibudilast (0.032 μm − 20 μm) potentiated isoprenaline‐induced cyclic AMP accumulation in a concentration‐dependent manner, being approximately 20 fold less potent than rolipram. Little or no effect on basal cyclic AMP levels was observed with either compound. The cyclic AMP‐dependent protein kinase activity ratio was significantly increased following incubation of eosinophils with either ibudilast (20 μm) or rolipram (20 μm) in the absence or presence of isoprenaline. 5 Leukotriene B4 (300 nm)‐induced thromboxane generation from guinea‐pig eosinophils was inhibited by ibudilast (IC50 = 11.3 ± 3.7 μm, n = 5) and rolipram (IC50 = 0.280 ± 0.067 μm, n = 5) in a concentration‐dependent manner. 6 Ibudilast (10 nm − 1 μm), whilst generally less potent than rolipram (1 nm − 1 μm), produced concentration‐dependent relaxation of spasmogen (methacholine, histamine, LTD4)‐induced tone in the guinea‐pig isolated tracheal strip. Ibudilast was less potent in reversing the methacholine (IC30 = 1.95 ± 0.40 μm, n = 6)‐induced contraction than those of histamine (IC50 = 0.18 ± 0.70 μm, n = 6) or leukotriene D4 (LTD4, IC50 = 0.12 ± 0.05 μm, n = 6). Rolipram also exhibited a similar pattern of activity, although the difference in potency against methacholine (IC50 = 0.1 ± 0.01 μm, n = 6) compared with the other two spasmogens, histamine (IC50 = 0.034 ± 0.017 μm, n = 7) and LTD4 (IC50 = 0.026 ± 0.008 μm, n = 7), was not as great. 7 These results demonstrate that ibudilast, like rolipram, has several biological actions on the eosinophil and airways smooth muscle which may be attributed to inhibition of cyclic AMP PDE. These actions may account, at least in part, for the recently reported anti‐asthma effects of ibudilast.

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].

A novel technique for the administration of bronchodilator drugs formulated as dry powders to the anaesthetized guinea pig.

A technique is described for the administration of dry powder formulations of bronchodilator drugs to the anaesthetized guinea pig. The technique has been evaluated by comparing the efficacy of several bronchodilator drugs administered either as dry powders coformulated with a lactose carrier or as nebulized solutions. In each case, the dry powder formulation had comparable bronchodilator activity and duration of action to an equivalent dose administered as a nebulized solution. This technique offers a simple and inexpensive method for the rapid screening of bronchodilator drugs without the problems associated with aerosol administration.