Gordon Dent

The effect of selective and non-selective phosphodiesterase inhibitors on allergen- and leukotriene C4-induced contractions in passively sensitized human airways

Non‐selective inhibitors of cyclic nucleotide phosphodiesterase (PDE) block allergen‐induced contraction of passively sensitized human airways in vitro by a dual mechanism involving a direct relaxant effect on smooth muscle and inhibition of histamine and cysteinyl leukotriene (LT) release from airways. We investigated the effects of non‐selective PDE inhibitors and selective inhibitors of PDE3 and PDE4 in order to determine the involvement of PDE isoenzymes in the suppression of allergic bronchoconstriction. Macroscopically normal airways from 76 patients were sensitized with IgE‐rich sera (>250 u ml−1) containing specific antibodies against allergen (Dermatophagoides farinae). Contractile responses of bronchial rings were assessed using standard organ bath techniques. Passive sensitization caused increased contractile responses to allergen, histamine and LTC4. Non‐selective PDE inhibitors (theophylline, 3‐isobutyl‐1‐methylxanthine [IBMX]), a PDE3‐selective inhibitor (motapizone), PDE4‐selective inhibitors (RP73401, rolipram, AWD 12‐281) and a mixed PDE3/4 inhibitor (zardaverine) all significantly relaxed inherent bronchial tone at resting tension and to a similar degree. Theophylline, IBMX, zardaverine and the combination of motapizone and RP73401 inhibited the contractile responses to allergen and LTC4. Pre‐treatment with motapizone, RP73401, rolipram or the methylxanthine adenosine receptor antagonist, 8‐phenyltheophylline, did not significantly decrease responses to either allergen or LTC4. We conclude that combined inhibition of PDE3 and PDE4, but not selective inhibition of either isoenzyme or antagonism of adenosine receptors, is effective in suppressing allergen‐induced contractions of passively sensitized human airways. The relationship between allergen‐ and LTC4‐induced responses suggests that PDE inhibitors with PDE3 and PDE4 selectivity are likely to act in part through inhibition of mediator release and not simply through direct relaxant actions on airway smooth muscle.

Selective phosphodiesterase inhibitors for the treatment of bronchial asthma and chronic obstructive pulmonary disease.

Theophylline is commonly used in the treatment of obstructive airway diseases. The identification and functional characterization of different phosphodiesterase (PDE) isoenzymes has led to the development of various isoenzyme‐selective inhibitors as potential anti‐asthma drugs. Considering the distribution of isoenzymes in target tissues, with high activity of PDE3 and PDE4 in airway smooth muscle and inflammatory cells, selective inhibitors of these isoenzymes may add to the therapy of chronic airflow obstruction. However, initial data from clinical trials with selective PDE3 and PDE4 inhibitors have been somewhat disappointing and have tempered the expectations considerably since these drugs had limited efficacy and their use was clinically limited through side effects. The improved understanding of the molecular biology of PDEs enabled the synthesis of novel drugs with an improved risk/benefit ratio. These ‘second generation’ selective drugs have produced more promising clinical results not only for the treatment of bronchial asthma but also for the treatment of chronic obstructive pulmonary disease.

Radioligand binding of antagonists of platelet-activating factor to intact human platelets

Two new antagonists of platelet‐activating factor (PAF), the pyrrolothiazole derivative 52770 RP and the triazolodiazepine WEB 2086, have been studied as radioligands in intact human platelets. [3H]52770 RP and [3H]WEB 2086 bound specifically to high‐affinity sites with dissociation constants (K d) of 14.8 and 6.1 nM, respectively. The maximal number of sites for [3H]52770 RP binding was approx. 15‐fold higher than for [3H]PAF and [3H]WEB 2086. In addition, C16‐PAF, lyso‐PAF, WEB 2086 and 52770 RP had K i values which were nearly identical for both [3H]PAF and [3H]WEB 2086, whereas only 52770 RP competed for [3H]52770 RP‐binding sites. These results demonstrate that in human platelets the sites of [3H]WEB 2086 binding are identical to [3H]PAF‐binding sites, whereas those of [3H]52770 RP are not. [3H]WEB 2086 appears, therefore, to be a suitable antagonist radioligand for labelling PAF receptors.

Prosurvival activity for airway neutrophils in severe asthma

Background Airway neutrophilia is a recognised feature of chronic severe asthma, but the mechanisms that underlie this phenomenon are unknown. Evidence for factors present in airway secretions that prolong neutrophil survival has been sought and it has been hypothesised that these might be augmented in neutrophilic asthma. Methods Non-smoking subjects with severe asthma (SA) or mild asthma (MA) and healthy control subjects (HC) underwent sputum induction. The SA group was subdivided into subjects with neutrophil counts above (SA-high) and those within the normal range (SA-low). Apoptotic neutrophils were enumerated in the cellular phase while the fluid phase was assessed for its ability to prolong the in vitro survival of blood-derived neutrophils using morphometric and flow cytometric analyses. Results There was a significant difference between all four subject groups with respect to the percentage of apoptotic sputum neutrophils (Kruskal–Wallis, p=0.042). Cuzick test showed a highly significant (p=0.008) trend towards decreasing numbers of apoptotic neutrophils across the four groups with increasing asthma severity and neutrophil count. The sputum antiapoptotic activity was also different between the groups (p=0.039), with a highly significant (p=0.005) decreasing trend across the four groups. The survival effect could not be inhibited by blocking selective chemotaxin receptors, neutralising neutrophil survival factors, inhibiting phosphatidylinositol-3-kinase (using LY294002) or with pertussis toxin pretreatment. Similarly, it could not be explained by lipopolysaccharide contamination or by the presence of inhaled corticosteroids in sputum. Conclusions These data demonstrate the capacity of as yet unidentified factor(s) in the airways of subjects with asthma to delay human neutrophil apoptosis and extend their lifespan as a potential mechanism contributing to unresolving airways neutrophilia in severe asthma.

Salmeterol is a competitive antagonist at β-adrenoceptors mediating inhibition of respiratory burst in guinea-pig eosinophils

The ability of the long-acting beta-adrenoceptor agonists eformoterol and salmeterol to inhibit leukotriene (LT) B4 (100 nM; approximately EC70)-induced hydrogen peroxide (H2O2) generation by guinea-pig peritoneal eosinophils was investigated and compared with salbutamol. Eformoterol and salbutamol produced a concentration-dependent inhibition of LTB4-induced H2O2 generation with pIC50 values of 6.22 and > 5.0 respectively. The inhibitory effect eformoterol was mediated through an interaction with beta-adrenoceptors for it was antagonised by propranolol with an affinity (7.21) that was independent of antagonist concentration (100 nM and 1 microM). In contrast, salmeterol (1 nM to 10 microM) failed to inhibit H2O2 generation at any concentration examined irrespective of the pre-incubation time (0, 0.25, 0.5, 1, 2, 15 or 30 min). Salmeterol did, however, competitively antagonise (slope of Schild plot = 0.91) the inhibition of H2O2 generation induced by eformoterol with a pA2 of 5.9. Possible explanations for the lack of inhibitory effect of salmeterol on LTB4-induced respiratory burst are advanced and critically discussed.

Effects of salbutamol on bronchoconstriction, bronchial hyperresponsiveness, and leucocyte responses induced by platelet activating factor in man.

Platelet activating factor, a potent mediator of inflammation, causes a sustained increase in airway responsiveness to methacholine in man and has been implicated in asthma. The effect of the beta 2 agonist salbutamol (200 micrograms by inhalation) on platelet activating factor induced bronchoconstriction and airway hyperresponsiveness was studied in seven normal subjects in a double blind, crossover study. Salbutamol only partially inhibited the platelet activating factor induced fall in partial flow at 30% of vital capacity (Vp30) (mean percentage fall 47.6 (SEM 7.9); p less than 0.001), whereas it completely blocked a similar degree of bronchoconstriction induced by methacholine. Salbutamol did not prevent the accompanying transient flushing and chest irritation and did not affect the transient neutropenia (mean % fall 69.5 (13.6); p less than 0.01) or the rebound neutrophilia (mean % increase 84.7 (24.7); p less than 0.05) that followed platelet activating factor. There was an increase in the airway responsiveness to methacholine following inhalation of platelet activating factor, the maximum mean change being a three fold increase in PC40 (the provocative concentration of methacholine causing a 40% fall in Vp30) on day 3 (p less than 0.01). Salbutamol caused a significant attenuation of this response on day 3 (p less than 0.02) but had no significant effect on days 1 and 7. Thus a therapeutic dose of salbutamol caused partial inhibition of platelet activating factor induced bronchoconstriction and had a minimal effect on the increased bronchial responsiveness following platelet activating factor.

Effect of a Paf antagonist, WEB 2086, on airway microvascular leakage in the guinea-pig and platelet aggregation in man.

1 The triazolodiazepine WEB 2086 has been evaluated as an antagonist of platelet‐activating factor (Paf) by studying its effects on Paf‐induced human platelet aggregation and microvascular leakage in guinea‐pigs. 2 WEB 2086 inhibited Paf‐induced platelet aggregation in platelet‐rich plasma in vitro (IC50 = 117 ± 35 nm, mean ± s.d.) but had no effect on adenosine 3′,5′‐diphosphate‐induced aggregation. 3 Paf‐induced microvascular leakage, measured by the extravasation of intravenously‐injected Evans blue dye, was inhibited in a dose‐related fashion in the airways and other tissues by WEB 2086, achieving a maximal inhibitory effect at 10 μg kg−1, i.v. 4 However, WEB 2086 (10 μg kg−1, i.v.) did not inhibit a comparable increase in vascular permeability induced by ovalbumin in sensitized guinea‐pigs. 5 We conclude that WEB 2086 is a potent antagonist of Paf and that Paf does not appear to be responsible for antigen‐induced microvascular leakage.

Evidence regarding the utility of multiple mini-interview (MMI) for selection to undergraduate health programs: A BEME systematic review: BEME Guide No. 37

Abstract Background: In the 11 years since its development at McMaster University Medical School, the multiple mini-interview (MMI) has become a popular selection tool. We aimed to systematically explore, analyze and synthesize the evidence regarding MMIs for selection to undergraduate health programs. Methods: The review protocol was peer-reviewed and prospectively registered with the Best Evidence Medical Education (BEME) collaboration. Thirteen databases were searched through 34 terms and their Boolean combinations. Seven key journals were hand-searched since 2004. The reference sections of all included studies were screened. Studies meeting the inclusion criteria were coded independently by two reviewers using a modified BEME coding sheet. Extracted data were synthesized through narrative synthesis. Results: A total of 4338 citations were identified and screened, resulting in 41 papers that met inclusion criteria. Thirty-two studies report data for selection to medicine, six for dentistry, three for veterinary medicine, one for pharmacy, one for nursing, one for rehabilitation, and one for health science. Five studies investigated selection to more than one profession. MMIs used for selection to undergraduate health programs appear to have reasonable feasibility, acceptability, validity, and reliability. Reliability is optimized by including 7–12 stations, each with one examiner. The evidence is stronger for face validity, with more research needed to explore content validity and predictive validity. In published studies, MMIs do not appear biased against applicants on the basis of age, gender, or socio-economic status. However, applicants of certain ethnic and social backgrounds did less well in a very small number of published studies. Performance on MMIs does not correlate strongly with other measures of noncognitive attributes, such as personality inventories and measures of emotional intelligence. Discussion: MMI does not automatically mean a more reliable selection process but it can do, if carefully designed. Effective MMIs require careful identification of the noncognitive attributes sought by the program and institution. Attention needs to be given to the number of stations, the blueprint and examiner training. Conclusion: More work is required on MMIs as they may disadvantage groups of certain ethnic or social backgrounds. There is a compelling argument for multi-institutional studies to investigate areas such as the relationship of MMI content to curriculum domains, graduate outcomes, and social missions; relationships of applicants’ performance on different MMIs; bias in selecting applicants of minority groups; and the long-term outcomes appropriate for studies of predictive validity.

Differential dependence of eosinophil chemotactic responses on phosphoinositide 3-kinase (PI3K)

Background:  Control of eosinophil migration to sites of inflammatory responses is a potentially therapeutic intervention in diseases such as bronchial asthma. Chemoattractants, their receptors and the associated signalling pathways may, therefore, be important targets for novel therapeutics. While several potentially important chemoattractants have been identified, the signalling pathways mediating their actions are incompletely understood.

Selective Phosphodiesterase Inhibitors in the Therapy of Asthma

Cyclic nucleotide phosphodiesterases form a group of enzymes that catalyse the breakdown of the intracellular second messengers cyclic AMP and cyclic GMP. Inhibitors of these enzymes, such as theophylline and enprofylline, are standard agents in the therapy of bronchial asthma but are limited in their usefulness by their poor potency and frequent adverse effects. An extensive research effort in recent years has identified 7 families of phosphodiesterase, comprising more than 33 separate isoenzymes, that may represent targets for novel anti-asthma therapies. The role of cyclic AMP and cyclic GMP in the regulation of cell function in many airway tissues and immune cells involved in the pathophysiology of asthma has been identified, and the pharmacological actions of isoenzyme-selective phosphodiesterase inhibitors upon these cells are under investigation. Although the first target for investigation was the airway smooth muscle, whose episodic constriction is the primary sign of asthma, increasing interest is developing in the ability of phosphodiesterase inhibitors to modulate the activity of inflammatory cells and, thereby, to modify the underlying pathological processes of the disease. Recent advances in the understanding of the actions of phosphodiesterase inhibitors on T lymphocytes and eosinophils are particularly exciting, and may indicate a place for these drugs in a new generation of treatments for asthma.