Lazzaro Mazzoni

Pulmonary edema induced by allergen challenge in the rat: noninvasive assessment by magnetic resonance imaging.

The course of pulmonary edema formation after an intratracheal (i.t.) instillation of ovalbumin was followed noninvasively by magnetic resonance imaging ( MRI ) in actively sensitized Brown Norway (BN) rats. Changes in edema volume assessed by MRI mimicked the results from the analysis of the number and activation of inflammatory cells recovered from the broncho‐alveolar lavage (BAL) fluid. Rats treated with budesonide did not develop edema following challenge with ovalbumin, and these animals showed a significant decrease in BAL fluid inflammatory cell numbers and eosinophil peroxidase and myeloperoxidase activities. Thus, following lung edema formation by MRI provides a reliable means of assessing pulmonary inflammation after allergen challenge. Unlike BAL fluid analysis, which requires killing animals at each time point, this method is noninvasive. MRI could be of importance for the noninvasive profiling of anti‐inflammatory drugs in animal models of asthma and in the clinic. Magn Reson Med 45:88–95, 2001.

Mechanism of airway hyperresponsiveness to adenosine induced by allergen challenge in actively sensitized Brown Norway rats

We have explored the role of allergen sensitization and challenge in defining the response of the airways of the Brown Norway (BN) rat to adenosine. In naïve animals or in rats sensitized to ovalbumin (OA) adenosine induced only weak bronchoconstrictor responses. Challenge of sensitized animals with OA induced a marked airway hyperresponsiveness to adenosine which was not seen with methacholine or bradykinin. The augmented bronchoconstrictor response to adenosine was not affected by acute bivagotomy or atropine nor mimicked by an i.v. injection of capsaicin. It was, however, blocked selectively by disodium cromoglycate methysergide or ketanserin and reduced in animals treated sub‐chronically with compound 48/80. The augmented response to adenosine was associated with increases in the plasma concentrations of both histamine and 5‐hydroxytryptamine (5‐HT), which were attenuated by pretreatment with disodium cromoglycate, and degranulation of mast cells in the lung. Parenchymal strips from lungs removed from sensitized rats challenged with OA gave augmented bronchoconstrictor responses to adenosine relative to strips from sensitized animals challenged with saline. Responses were inhibited by methysergide and disodium cromoglycate. These data demonstrate a marked augmentation of the bronchoconstrictor response to adenosine in actively sensitized BN rats challenged with OA. The augmented response is primarily a consequence of mast cell activation, leading to the release of 5‐HT, which in turn induces bronchoconstriction. Our data further suggest the involvement of a discrete lung‐based population of mast cells containing and releasing mainly 5‐HT and brought into play by prior exposure to allergen.

Resolution of the oedema associated with allergic pulmonary inflammation in rats assessed noninvasively by magnetic resonance imaging.

Magnetic resonance imaging (MRI) was used to study noninvasively the effects of compounds to resolve inflammation induced by ovalbumin (OVA) challenge in the lungs of actively sensitised rats. Marked oedematous signals were detected between 24 and 96 h following OVA in vehicle‐treated animals. When administered 24 h after OVA, budesonide, a glucocorticosteroid, or 4‐(8‐benzo[1,2,5]oxadiazol‐5‐yl‐[1,7]naphthyridin‐6‐yl)‐benzoic acid (NVP‐ABE171), a selective phosphodiesterase 4 inhibitor, increased the rate of resolution of established oedematous signals detected by MRI. The effect was evident 3 h after drug administration and the signals were nearly fully resolved at 96 h postchallenge. The drug‐induced rapid resolution of MRI signals was not accompanied by changes in parameters of inflammation in the bronchoalveolar lavage fluid, but was associated with perivascular oedema detected histologically. In conclusion, the effects of anti‐inflammatory drugs on a component of allergic inflammation can be monitored by following with MRI the rate of resolution of the associated oedematous signals.

β-adrenergic tachyphylaxis in the rat and its reversal and prevention by ketotifen

A strong, long-lasting and reproducible tachyphylaxis was produced in rats by implantation of osmotic minipumps delivering isoprenaline continuously. The degree of tachyphylaxis was determined by measuring the inhibitory effect of isoprenaline on the passive cutaneous anaphylaxis (PCA). One dose of ketotifen given 1 h before the PCA test reversed this in vivo tachyphylaxis, as did dexamethasone given 24 h earlier. Implantation of a second minipump containing ketotifen prevented the development of tachyphylaxis. Weak tachyphylaxis was induced in guinea-pig trachea in vitro by incubation with a high concentration of isoprenaline, the effect being estimated by measuring the relaxation of carbachol-contracted trachea. Ketotifen partially restored the sensitivity of the trachea but this was considered to be a direct potentiation of isoprenaline effects rather than a reversal of tachyphylaxis since the same effect was seen in non-pretreated trachea. It is thought that the reversal of experimental beta-adrenergic tachyphylaxis by ketotifen could have implications for its use in the prophylactic treatment of asthma.

Evidence for an atypical receptor mediating the augmented bronchoconstrictor response to adenosine induced by allergen challenge in actively sensitized Brown Norway rats

The bronchoconstrictor response to adenosine is markedly and selectively increased following ovalbumin (OA) challenge in actively sensitized, Brown Norway rats. We present a pharmacological analysis of the receptor mediating this response. Like adenosine, the broad‐spectrum adenosine receptor agonist, NECA, induced dose‐related bronchoconstriction in actively sensitized, OA‐challenged animals. In contrast, CPA, CGS 21680 and 2‐Cl‐IB‐MECA, agonists selective for A1 A2A and A3 receptors, respectively, induced no, or minimal, bronchoconstriction. Neither the selective A1 receptor antagonist, DPCPX, nor the selective A2A receptor antagonist, ZM 241385, blocked the bronchoconstrictor response to adenosine. MRS 1754, which has similar affinity for rat A2B and A1 receptors, failed to block the bronchoconstrictor response to adenosine despite blockade of the A1 receptor‐mediated bradycardia induced by NECA. 8‐SPT and CGS 15943, antagonists at A1, A2A, and A2B but not A3 receptors, inhibited the bronchoconstrictor response to adenosine. However, the degree of blockade (approximately 3 fold) did not reflect the plasma concentrations, which were 139 and 21 times greater than the KB value at the rat A2B receptor, respectively. Adenosine and NECA, but not CPA, CGS 21680 or 2‐Cl‐IB‐MECA, induced contraction of parenchymal strip preparations from actively sensitized OA‐challenged animals. Responses to adenosine could not be antagonized by 8‐SPT or MRS 1754 at concentrations >50 times their affinities at the rat A2B receptor. The receptor mediating the bronchoconstrictor response to adenosine augmented following allergen challenge in actively sensitized BN rats cannot be categorized as one of the four recognized adenosine receptor subtypes.

Effects of wortmannin on airways inflammation induced by allergen in actively sensitised Brown Norway rats

We have investigated the effect of wortmannin, a potent and selective inhibitor of phosphatidylinositol-3-kinase, on the immediate-type allergic response and the late phase pulmonary inflammation induced by allergen challenge in the ovalbumin-sensitised Brown Norway rat. Intratracheal (i.t.) instillation of ovalbumin induced dose-related bronchoconstrictor responses. Administration of wortmannin (1, 10 or 100 microg kg(-1) i.t., 1 h prior to challenge) induced a marked and dose-dependent inhibition of ovalbumin-induced bronchospasm (ED(50) ca. 5 microg kg(-1) i.t.). At similar doses, wortmannin also suppressed the bronchoconstrictor responses to 5-hydroxytryptamine and methacholine but the degree of blockade of these spasmogens (1.4-1.9-fold) was less than that of ovalbumin (>20-fold). Wortmannin, given intratracheally 1 h prior to allergen challenge, also suppressed the increases in bronchoalveolar lavage fluid leukocyte numbers and eosinophil peroxidase activity measured 24 h post challenge. However, relatively high doses were necessary (ED(50) ca. 100 microg kg(-1) i.t.). The potency of wortmannin was increased when dosed 1 h prior to and 24 h after allergen challenge and the readout was 48 h after challenge (ED(50) 3-5 microg kg(-1) i.t.). Thus, wortmannin is a potent inhibitor of the bronchoconstrictor response induced by allergen in the airways of actively sensitised Brown Norway rats. Inhibition of phosphatidylinositol-3-kinase, an obligatory step in mast cell activation in response to allergen, is the presumed mechanism of action. The fact that similar doses of wortmannin do not suppress the late response to allergen suggests a minimal role for the mast cell in generating the late response to allergen in this model. The striking increase in potency to inhibit the late response when dosed 1 h prior to and 24 h after allergen challenge with the readout taken at 48 h may represent an effect of wortmannin to suppress the migration of leukocytes.

Airway hyperresponsiveness to adenosine induced by lipopolysaccharide in Brown Norway rats

We have explored the effects of bacterial endotoxin (lipopolysaccharide; LPS) on the response of the airways of Brown Norway (BN) rats to adenosine. Comparisons have been drawn with the effects on responses to methacholine and 5‐hydroxytryptamine. In vehicle‐challenged animals, adenosine, given i.v. was only a weak bronchoconstrictor. In contrast, 1 h following intratracheal administration of LPS, 0.3 mg kg−1, bronchoconstrictor responses to adenosine were markedly and selectively enhanced. At this time point, there were no significant changes in leukocyte numbers, eosinophil peroxidase and myeloperoxidase activities or protein concentrations in bronchoalveolar lavage (BAL) fluid. Twenty‐four hours after challenge, the sensitivity of the airways to both adenosine and methacholine was reduced relative to the earlier time point and there were substantial increases in each marker of inflammation in BAL fluid. The bronchoconstrictor response to adenosine was blocked selectively by methysergide, disodium cromoglycate and the broad‐spectrum adenosine receptor antagonist, 8‐SPT, but not by DPCPX or ZM 243185, selective antagonists for the A1 and A2A receptors, respectively. Thus, the response to adenosine augmented following LPS is mast cell mediated and involves a receptor which can be blocked by 8‐SPT but not by selective A1 or A2A receptor antagonists. It thus bears similarity to the augmented response to adenosine induced by allergen challenge in actively sensitized BN rats. Exposure to LPS could be a factor along with allergen in determining the increased sensitivity of the airways of asthmatics to adenosine.

Disease activated drugs: a new concept for the treatment of asthma

Disease activated drugs (DAD) are pro-drugs of one active principle or combinations of two drugs, which have a proven efficacy for the treatment of the target disease. In opposition to pro-drugs, DAD are activated in inflamed but not normal tissues. Due to the disease specific activation, the amount of locally released drug(s) should be related directly to the severity of the inflammation. To test this concept in asthma a PDE4 inhibitor, an isoquinoline derivative, was chemically derivatized into pro-drugs or combined with corticosteroids. These new compounds were more readily cleaved into active PDE4 inhibitor, in bronchoalveolar lavage fluid (BALF) from Brown-Norway rats with lung inflammation than in BALF from rats without airway inflammation. The DAD concept (local selective release and improved therapeutic window) was validated in vivo using the inhibition of methacholine induced bronchoconstriction in guinea pigs with or without ozone induced lung inflammation. An example of DAD hydrolysis (isoquinoline-dexamethasone) was also examined in BALF from asthmatics and healthy volunteers.

Role of endogenous adenosine in the acute and late response to allergen challenge in actively sensitized Brown Norway rats

Endogenous adenosine has been suggested to amplify the response of airway mast cells to allergen in vivo. We have sought evidence for this by monitoring the acute and late‐phase response to allergen in Brown Norway (BN) rats actively sensitised to ovalbumin (OA) and treated either with adenosine deaminase (ADA) linked covalently to polyethylene glycol (PEG‐ADA; Adagen®) to decrease adenosine availability or with erythro‐9‐(2‐hydroxy‐3‐nonyl)adenine (EHNA), an inhibitor of ADA, plus S‐(4‐nitrobenzyl)‐6‐thioinosine (NBTI), an inhibitor of facilitated adenosine transport, to increase adenosine availability. The cardiovascular effects of adenosine (0.01–3 mg kg−1 i.v.) were significantly reduced in PEG‐ADA‐treated animals and augmented in EHNA/NBTI‐treated animals. The difference in sensitivity to adenosine in the treated groups was 33‐ and 15‐fold, at the level of 30% reduction in blood pressure and heart rate, respectively. The acute response to allergen, given either intravenously or intratracheally, was quantified as bronchoconstriction. The late phase to allergen was measured as the influx and activation of immunoinflammatory cells into the bronchoalveolar lavage fluid 24 h after challenge. Despite evidence of a substantial difference in adenosine availability following pretreatment with PEG‐ADA or EHNA/NBTI, there were no differences in either the acute or late response to allergen in the actively sensitised BN rat. Our data suggest no role for endogenous adenosine in determining the response to allergen under our experimental conditions.