Ivan M. Richards

Contribution of Leukotriene B4 to Airway Inflammation and the Effect of Antagonists

Inhalation of aerosols of ovalbumin in sensitized guinea pigs produced a marked, bronchoalveolar eosinophilia 24 hr after challenge. The lung eosinophilia was not prevented by the cyclooxygenase inhibitors, indomethacin or PAF antagonists (WEB-2086 and L-652731) but was inhibited by methylprednisolone, the 5-LO inhibitor, U-66858 and a series of structural analogs of LTB4, U-75302, U-77692, U-75485 and U-78489. The effectiveness of LTB4 antagonists but not PAF antagonists in vivo was consistent with in vitro studies in which LTB4 was shown to be far more chemotactic than PAF for guinea pig eosinophils. LTB4 elicited maximal directional migration of guinea pig eosinophils at concentrations from 10(-7) M to 10(-9) M while PAF showed no effect over the same concentration range. The structural analogs of LTB4 were shown to inhibit LTB4 induced chemotaxis of guinea pig eosinophils and produced a dose-related inhibition of binding of LTB4 to guinea pig eosinophil membranes. To add further proof to the hypothesis that LTB4 contributed to the antigen-induced lung eosinophilia we attempted to measure LTB4 release into BAL fluid immediately after and at various time points up to 24 hr after antigen inhalation. However, using a sensitive radioimmunoassay (detection limit 10 pg/ml) very low levels of LTB4 (24.9-67.9 pg/ml) or its metabolite, 20-OH LTB4 (24.9-98.2 pg/ml) were detected in BAL fluid and these levels did not increase significantly following antigen provocation. Inhalation of LTB4 aerosols in unsensitized Brown-Norway rats or inhalation of aerosols of ovalbumin in sensitized Brown-Norway rats also produced a marked late-phase eosinophil-rich influx of inflammatory cells into the lungs. The lung eosinophilia in the rat was prevented by two structurally unrelated leukotriene B4 (LTB4) antagonists, U-75302 and Ly255283. These data implicate LTB4 as a mediator of allergen-induced bronchopulmonary eosinophilia. Leukotriene B4 antagonists may provide leads for the development of compounds which inhibit the chronic airway inflammation associated with asthma in man.

Novel steroid‐based inhibitors of lung inflammation

It is now well-recognized that eosinophil products contribute to the mucosal damage which is a prominent feature of bronchial asthma. Eosinophils migrate to the bronchial epithelium following allergen inhalation in sensitive subjects, and increases in eosinophil numbers are associated with late-phase broncho-conslrictions [I]. The eosinophils capacity to generate lipid mediators [2.3] and the presence of cytotoxic. highly-charged, arginine-rich proteins in the eosinophil granules implicate this cell further as a contributor to mucosal inflammation. An attractive goal in the search for novel anti-asthma drugs would be to discover and develop compounds which inhibit eosinophil activation and/or prevent the influx of eosinophils into the airways. Steroids arc probably the most efficacious drugs currently available for the treatment of asthma, and improvements in symptoms in patients treated with steroids have been associated with a parallel reduction in blood eosinophil counts [4]. Corticosteroids have also been shown to abrogate the development of late-phase asthmatic reactions with a parallel reduction in eosinophil activity as measured by serum eosinophil cationic protein (ECP) levels [5]. Inhaled glucocorticoids which demonstrate few side-effects are increasingly being used worldwide as a prophylactic treatment for the disease. Novel inhaled glucocorticoids which have been designed to break down rapidly after reaching the systemic circulation are currently being developed and are even less likely to suppress the hypothalamo-pituitary adrenal axis. In this paper we have reviewed some of the novel. steroid-based molecules currently in clinical development for treating asthma. In addition we emphasize the change in direction the pharmaceutical industry has taken towards the use of inflammation-based animal models for the screening of novel compounds, and we also describe the activity of some novel, non-glucocorticoid. 21 -aminosteroids which may prove useful in the treatment of asthma.

Biological activity of leukotriene B4 analogs: Inhibition of guinea pig eosinophil migration in vitro by the 2,6-disubstituted pyridine analogs U-75,302 and U-75,485

A late phase antigen-induced bronchoalveolar eosinophilia has been demonstrated in ovalbumin sensitized guinea pigs (1,2). This in vivo response to antigen inhalation can be inhibited by a 2,6-disubstituted pyridine analog of LTB4, U-75,302(2) (3). In the present study, the mechanism of the drug action was studied by assessing the activity of U-75,302 and a second analog, U-75,485 to displace [3H]-leukotriene B4 binding at the guinea pig eosinophil membrane, as well as their action as chemoattractants or inhibitors of the directional migration of guinea pig eosinophils in vitro. Radioligand competition experiments demonstrated that both analogs interacted strongly with the high affinity LTB4 binding sites on guinea pig eosinophil membrane. Both analogs are powerful chemoattractants for guinea pig eosinophils since they induced directional migration of guinea pig eosinophils when administered alone. In addition, when the cells were treated with either analog and their chemotaxis response was measured in response to a natural chemoattractant, both U-75,302 and U-75,485 at concentrations of 0.1 to 100 microM dose dependently inhibited the LTB4 induced chemotaxis response. The EC50s obtained for U-75,302 and U-75,485 as inhibitors of LTB4 induced guinea pig eosinophil chemotaxis were estimated to be 11.5 +/- 5.5 microM and 5.4 +/- 2.5 microM respectively. Under the same conditions, they had no significant effect upon eosinophil migration induced by zymosan activated plasma at concentrations below 100 microM. We suggest that the inhibition of antigen-induced eosinophil infiltration in guinea pig airway in vivo by U-75,302 or U-75,485 may be a result of partial antagonism or desensitization at the LTB4 receptor level of guinea pig eosinophils.

Effect of the 21-aminosteroid, U-74389F, on hyperoxic lung injury in rats

Hyperoxia (>95% oxygen) in rats caused an increase in lung weight and an accumulation of fluid in the thorax. The mean lung wet weight of air-breathing controls at 60 h was 1.2±0.01 g, and that of vehicle-treated, oxygen-exposed animals was 2.45±0.05 g. Treatment with the 21-aminosteroid U-74389F, 3, 10, and 30 mg/kg twice daily throughout oxygen exposure, produced 8, 42, and 18% inhibition of the oxygen-induced increase in lung weight, respectively. However, U-74389F did not inhibit the hyperoxia-induced accumulation of neutrophils in bronchoalveolar lavage fluid.No pleural fluid could be aspirated from the thorax of air-breathing controls. The volume of pleural fluid in oxygen-exposed, vehicle-treated animals and animals treated with 3, 10, and 30 mg/kg U-74389F b.i.d. was 6.5±0.9, 2.6±0.6, 0.8±0.3, and 1.3±0.5 ml, respectively.U-74389F or its biologs are of potential value for the treatment of lung diseases in which oxidant damage has been implicated.

Chasing the elusive animal model of late-phase bronchoconstriction: Studies in dogs, guinea pigs and rats

SummaryAntigen inhalation in sensitized dogs, guinea pigs and rats resulted in a marked, late-phase, eosinophil-rich, influx of inflammatory cells into the bronchial lumen. Attempts to demonstrate an associated late-phase bronchoconstriction were disappointing. We were unable to demonstrate a late-phase bronchoconstriction in either rats or dogs, even when dogs were pretreated with metyrapone to reduce blood cortisol levels. In ovalbumin-sensitized guinea pigs, challenged with low doses of ovalbumin, we observed an immediate bronchoconstriction, a late-phase bronchopulmonary eosinophilia but no late-phase bronchoconstriction. However, inhalation of very high doses of antigen in mepyramine-treated sensitized guinea pigs did induce a moderate late-phase bronchoconstriction.

Inhibition of the Presumably Leukotriene-Dependent Component of Antigen-Induced Bronchoconstriction in the Guinea Pig by Piriprost (U-60,257)

Piriprost (U-60,257), a selective inhibitor of the 5-lipoxygenase, inhibited bronchoconstriction in a modification of the Konzett-Rossler procedure in which bronchoconstriction in sensitized guinea pigs may reflect leukotriene production. The duration of action of this compound and also the possible development of tachyphylaxis to its action upon repeated dosing were studied using a newly developed procedure for dosing conscious animals via the aerosol route. Duration of action with the lowest concentration which afforded maximum protection (180 s exposure to a 0.01% solution) was approximately 1.5 h. No decrease in drug potency was observed after 7 daily drug treatments.