Proteasome inhibition: A novel mechanism to combat asthma.

BACKGROUND: Nuclear factor-kappaB (NF-kappaB) is a critical transcription factor required for the regulation of many genes involved in inflammatory responses to noxious stimuli. On activation, NF-kappaB induces the transcription of numerous proinflammatory cytokines, enzymes, and cellular adhesion molecules. Blockade of the proteasome with selective inhibitors attenuates the effects of NF-kappaB, leading to suppression of the inflammatory response. OBJECTIVE: We sought to determine whether proteasome inhibitors would be active in a model of asthma. METHODS: The mouse delayed-type hypersensitivity model was used to screen a panel of compounds for in vivo activity. The proteasome inhibitor, PS-519, was shown to be the most active in this model and was selected for further development. Allergen-induced pulmonary eosinophilia in Brown Norway rats was used subsequently to determine anti-inflammatory activity in an animal model. RESULTS: Direct administration of PS-519 into the lungs significantly reduced leukocyte numbers, particularly the selective increase in eosinophils. Because steroids are the mainstay anti-inflammatory therapy in asthma, and data is available to suggest their possible interaction to suppress the activation of NF-kappaB, rats were also treated by inhalation with combinations of a steroid and the proteasome inhibitor. In both the delayed-type hypersensitivity and the animal eosinophil model, low doses of proteasome inhibitors were shown to be effective when given with low doses of steroids. CONCLUSION: Taken together, the present data suggest that proteasome inhibition may represent a novel strategy for the treatment of inflammatory lung diseases such as asthma.

Effects of S-salbutamol on human isolated bronchus.

A range of stimuli have been used to determine the effect of S-salbutamol on contractile responses of human isolated bronchus. Significant augmentation of contraction was evident during responses to histamine or LTC4 but responses to EFS, methacholine, bradykinin and capsaicin were not influenced and allergic bronchospasm was significantly impaired by prior exposure to S-salbutamol. Since R-salbutamol relaxes human isolated bronchus, the capacity of S-salbutamol to enhance contractile responses to histamine or LTC4 cannot be attributed to activation of beta2-adrenoceptors. It is concluded, therefore, that these effects of S-salbutamol represent distinct pharmacological actions of the distomer. It is also suggested that the capacity of S-salbutamol to augment contraction of airway smooth muscle may contribute to hyperreactivity towards spasmogens when racemic salbutamol is used for symptom relief in asthma and chronic obstructive pulmonary disease (COPD).

Hyperresponsiveness of the airways following exposure of guinea-pigs to racemic mixtures and distomers of beta 2-selective sympathomimetics.

Allergic bronchospasm in sensitized guinea-pigs was totally suppressed by acute subcutaneous infusion of rac-salbutamol (0.69 microgram/kg per min) for < 1 h. More prolonged infusion of rac-salbutamol induced a progressive susceptibility to inhaled antigen so that, by 48 h, animals collapsed and died following inhalation of antigen. In anaesthetized animals, acute infusion of rac-salbutamol (1.67 micrograms/kg per min) suppressed airway obstruction, an effect that can be attributed to beta 2-adrenoceptor activation by the eutomer (R-salbutamol). Acute intravenous infusion of the distomer (S-salbutamol) (1.67 micrograms/kg per min) induced hyperresponsiveness to histamine without having any effect upon airway calibre. It is suggested therefore that subcutaneous infusion of rac-salbutamol initially abrogates the bronchoconstrictor response to antigen because the bronchodilator action of the eutomer predominates over hyperreactivity attributable to the distomer. Conversion from protection to susceptibility was not determined by reduced beta 2-adrenoceptor activation since animals could be protected from a lethal response to antigen by inhalation of rac-isoprenaline or by subcutaneous injection of rac-salbutamol. The seeming progressive loss of efficacy of R-salbutamol may result from disproportionate accumulation of S-salbutamol if, as in man, there is stereospecific metabolism of R-salbutamol. The capacity of S-salbutamol to evoke hyperresponsiveness is shared by S-isoprenaline and S-terbutaline and, as has been shown previously for rac-isoprenaline, the capacity of S-salbutamol to elicit hyperresponsiveness was not evidenced following section of the vagus nerves. No mechanism has yet been established which might account for this property of S-salbutamol or for other S-enantiomers of sympathomimetics.

Therapeutic potential of potassium channel openers in peripheral vascular disease and asthma.

Potassium channel opener's (KCOs) were originally thought of as nonselective smooth muscle relaxants. However, recent investigations in animal models of both peripheral vascular disease (PVD) and asthma have revealed interesting effects of these drugs as unexpectedly low doses. Hemodynamically, KCOs are interesting in PVD since they have little effect on blood supply to normally perfused skeletal muscle, but enhance perfusion to chronically ligated ischemic tissue. In animal PVD models, SDZ PCO-400 and cromakalim have been shown to improve recovery of muscle energy stores from ischemia or to preserve performance under conditions of ischemic contracture. Beneficial effects in rat PVD models were manifest at doses below those affecting systemic blood pressure and may be attributable to a selective dilatation of collateral vessels. With regard to the airways, the apparent efficacy of KCOs as antiasthmatic drugs seems not to be attributable solely to their bronchodilator activity. Although KCOs elicit no antiinflammatory effect in animal models, studies with SDZ PCO-400 in guinea pigs sensitized to antigen or treated with immune complexes have revealed that expression of airway hyperreactivity is significantly inhibited at drug doses exhibiting only modest bronchodilator activity. At least part of this action can be attributed to inhibition at the level of neural innervation of the airways, possibly through attenuation of nonadrenergic noncholinergic (NANC) transmission.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of a potassium channel opener (SDZ PCO 400) on guinea-pig and human pulmonary airways.

1. SDZ PCO 400 evoked dose-related relaxation of isolated airway smooth muscle. For human bronchus precontracted by endogenous tone or addition of carbachol (10(-5) M), IC50 values were 1.74 microM and 1.82 microM respectively. With guinea-pig trachea contracted by endogenous tone, a comparable IC50 (1.79 microM) was observed, but no IC50 (less than 100 microM) could be determined following contraction by carbachol (10(-6) M). 2. Airway obstruction induced by intravenous bombesin in the anaesthetized ventilated guinea-pig was diminished by intravenous injection of SDZ PCO 400 (ID50 54 micrograms kg-1) or by introduction into the duodenum (ID50 1.0 mg kg-1). Inhalation of nebulized SDZ PCO 400 (0.1 mg kg-1) diminished airway obstruction due to intravenous injection of histamine (3.2-5.6 micrograms kg-1) for up to 20 min. 3. Increased bronchoconstrictor responses to bombesin (180-240 ng kg-1) following intravenous infusion of platelet activating factor (PAF) or (+/-)-isoprenaline, or to histamine (1.0-3.2 micrograms kg-1) following intravenous injections of immune complexes, were suppressed following concomitant intravenous infusion of SDZ PCO 400 (ID50 0.3 mg kg-1 h-1, 1.0 mg kg-1 h-1 and 0.1 mg kg-1 h-1 respectively). 4. Intravenous injection of SDZ PCO 400 (0.1 mg kg-1) effected transient (less than 10 min) inhibition of histamine-induced bronchospasm, yet diminished, for prolonged periods [up to 40 min] the enhanced bronchoconstrictor responses to histamine that followed intravenous injections of immune complexes.The capacity of SDZ PCO 400 to resolve such established airway hyperreactivity was prevented by prior intraduodenal instillation of a potassium channel antagonist, glibenclamide (30 mg kg-').5. In sensitized guinea-pigs, SDZ PCO 400 inhaled as a dry powder (5.7 mg kg-') suppressed development of allergic airway hyperreactivity to histamine (1.8-3.2;pg kg-', i.v.), but failed to diminish accumulation of eosinophils or other inflammatory cells within the airway lumen 24 h after inhalation of ovalbumin.6. Preincubation (30 min) of isolated sensitized trachea of guinea-pig with SDZ PCO 400 (10-5-10-4M) did not influence contractile responses to ovalbumin. However in anaesthetized sensitized guinea-pigs,insufflation of SDZ PCO 400 (1.25 mg) as a powder substantially diminished airway obstruction that followed inhalation of ovalbumin. This effect was prevented by prior vagal section.7. It is concluded that SDZ PCO 400 reduces airway obstruction not only through direct actions on airway smooth muscle but also by impairing the expression of airway hyperreactivity, without directly influencing inflammatory events in the airways.

Development of airway hyperreactivity in the guinea-pig following allergic reactions.

It has been reported previously that acute allergic reactions in the anaesthetized guinea-pig do not evoke changed airway reactivity to histamine, animals sensitized to ovalbumin in aluminum hydroxide and exposed to allergen intravenously or by inhalation develop increased responsivity to histamine that persists for several hours. Animals that have been sensitized passively, by intravenous injection of antibody and exposed to allergen by intravenous injection, or which receive freshly prepared immune complexes by intravenous injection develop comparable airway reactivity.

Changes in airway sensitivity to histamine are not necessarily paralleled by changed sensitivity to acetylcholine.

In allergic asthmatics, it has been shown that airway sensitivity to histamine closely parallels sensitivity to methacholine, a finding which underlies the widely-held view that airway hyperreactivity of asthma is non-selective. We have been interested to evaluate the capacity of various agents to influence reactivity to intravenous injections of both histamine and acetylcholine in the anaesthetized guinea-pig. Intravenous infusion of PAF or (+/-)isoprenaline are procedures which increase airway reactivity to histamine, but leave reactivity to acetylcholine unaffected; by way of contrast, there is comparable enhancement of hyperreactivity to both histamine and acetylcholine following endotoxin infusion. These observations do not accord with the concept that obstruction of the airways per se accounts for changed reactivity of the airways and question the presumption that PAF might be a pivotal mediator of airway hyperreactivity in allergic asthma.

SDZ MKS 492.

SDZ MKS 492 (R(+)-(8-[( 1-(3,4-Dimethoxyphenyl)-2-hydroxyethyl)amino]-3,7-dihydro-7-(2- methoxyethyl)-1,3-dimethyl-1H-purine-2,6-dione) relaxes airway smooth muscle in vitro and impairs, or reverses, spasm of guinea-pig airways in vivo. Effects on bronchospasm in the guinea-pig may additionally include actions on airway hyperreactivity, since SDZ MKS 492 can inhibit the development, or expression, of airway hyperreactivity due to infusion of PAF, (+/-)isoprenaline, endotoxin or injection of immune complexes. In addition to influencing airway tone, inhaled SDZ MKS 492 diminishes the pulmonary accumulation of macrophages, eosinophils and neutrophils that follows inhalation of allergen by actively sensitized guinea-pigs. SDZ MKS 492 offers the prospect of a monotherapy for asthma, combining bronchodilator and anti-inflammatory activities with prophylactic efficacy.

Airway eosinophilia and airway hyperreactivity are parallel rather than sequential events in the guinea-pig.

It is known that eosinophil activation in the airways is associated with epithelial damage in both guinea-pigs and man and it has been presumed that airway hyperreactivity arises in consequence of these events. However, infusion of (+/- )isoprenaline induces airway hyperreactivity in the guinea-pig despite a reduction of eosinophil numbers in the airway lumen, whilst rh-GM-CSF or rh-IL3 induce eosinophilia of the airways without attendant airway hyperreactivity. These findings complement earlier studies which showed that airway hyperreactivity in sensitized animals reacting to inhaled allergen could not be correlated with the number of eosinophils in the airway lumen.

Actions of SDZ PCO 400 and cromakalim on airway smooth muscle in vivo.

SDZ PCO 400, and cromakalim are potassium channel opening drugs which relax airway smooth muscle and reverse airway obstruction due to intravenous administration of airway spasmogens. Reduction of airway obstruction by these drugs may be attributed to actions upon airway reactivity as well as airway smooth muscle spasmolysis; for, at concentrations which do not induce overt relaxation of airway smooth muscle, both SDZ PCO 400 and cromakalim suppressed development of airway hyperreactivity due to administration of immune complexes, PAF or (+/-)isoprenaline and reversed established airway hyperreactivity due to allergic reactions. When administered as aerosols prior to inhalation of allergen in actively sensitized guinea-pigs, or by the oral route in guinea-pigs with idiopathic eosinophilia, neither cromakalim nor SDZ PCO 400 influenced the influx of eosinophils into the airway lumen. Thus, SDZ PCO 400 and cromakalim may readily be distinguished from established prophylactic anti-asthma drugs such as cromoglicate and ketotifen. By influencing airway reactivity, potassium channel openers provide a novel approach to the resolution of airway obstruction in asthma.

Biochemical and electrical aspects of the tracheal relaxant action of AH 21-132.

In Triton X-100-skinned trachealis muscle, neither papaverine nor AH 21-132 modified responses to Ca2+. The (-)-enantiomer of AH 21-132 was more potent than the (+)-enantiomer both in relaxing intact trachealis muscle and in inhibiting tracheal cAMP phosphodiesterase (PDE). AH 21-132 (0.6 microM) potentiated forskolin in causing tracheal relaxation but did not potentiate isoprenaline, cromakalim or sodium nitrate. AH 21-132 (2 microM) potentiated all four agents in relaxing the trachea. AH 21-132 (1 microM) potentiated forskolin in increasing tissue cAMP content and, in higher concentration, itself increased tissue cAMP. Electrical effects of AH 21-132 included suppression of spontaneous slow waves and cellular hyperpolarisation. It is concluded that AH 21-132 lacks a direct depressant effect on the intracellular contractile machinery. The weight of evidence suggests that AH 21-132-induced relaxation results from inhibition of cAMP-PDE. However, in common with other PDE inhibitors. AH 21-132 increases tissue cAMP content only at concentration greater than that required to cause full relaxation.

The bronchodilator action of AH 21-132.

The benzonaphthyridine derivative, AH 21-132, has non-specific relaxant effects in isolated airways smooth muscle. The action of AH 21-132 in trachealis muscle is not antagonised by propranolol but AH 21-132 is slightly potentiated by epithelium removal. Electrophysiological recording from guinea-pig trachealis shows that AH 21-132-induced relaxation is accompanied by suppression of electrical slow waves and by cellular hyperpolarisation. Unlike theophylline, AH 21-132 does not cause spasm of cooled (12 degrees C), indomethacin-treated trachealis muscle, nor does it act as an antagonist at adenosine A1 receptors. AH 21-132 does not depress the Ca2+ sensitivity or responsiveness of Triton X-100 skinned trachealis fibres. In tracheal relaxant concentrations, AH 21-132 selectively inhibits cAMP phosphodiesterase (PDE) compared with cGMP-PDE. The (-)-enantiomer of AH 21-132 is more potent than its (+)-enantiomer both in causing tracheal relaxation and in inhibiting cAMP-PDE. When tested on PDE isoenzymes separated from bovine trachealis and guinea-pig cardiac ventricles, AH 21-132 exhibits selectivity as an inhibitor of the isoenzyme types III and IV. AH 21-132 increases the trachealis content of cAMP and cGMP, but only in concentration greater than that required fully to suppress the mechanical tone of the tissue. AH 21-132 has bronchodilator activity in anaesthetised, ventilated guinea-pigs when administered intraduodenally, intravenously or by inhalation. Inhaled AH 21-132 also provides bronchodilatation in healthy human volunteers in whom bronchoconstriction has been induced by inhaled methacholine.