Background: Leukotriene (LT) E-4 is the final active metabolite among the cysteinyl leukotrienes (CysLTs). Animal studies have identified a distinct LTE4 receptor, suggesting that current cysteinyl leukotriene type 1 (CysLT(1)) receptor antagonists can provide incomplete inhibition of CysLT responses. Objective: We tested this hypothesis by assessing the influence of the CysLT(1) antagonist montelukast on responses induced by means of inhalation of LTE4 in asthmatic patients. Methods: Fourteen patients with mild intermittent asthma and 2 patients with aspirin-exacerbated respiratory disease received 20 mg of montelukast twice daily and placebo for 5 to 7 days in a randomized, double-blind, crossover study (NCT01841164). The PD20 value was determined at the end of each treatment period based on an increasing dose challenge. Measurements included lipid mediators in urine and sputum cells 4 hours after LTE4 challenge. Results: Montelukast completely blocked LTE4-induced bronchoconstriction. Despite tolerating an at least 10 times higher dose of LTE4 after montelukast, there was no difference in the percentage of eosinophils in sputum. Urinary excretion of all major lipid mediators increased after LTE4 inhalation. Montelukast blocked release of the mast cell product prostaglandin (PG) D-2, as well as release of PGF(2 alpha) and thromboxane (Tx) A(2), but not increased excretion of PGE(2) and its metabolites or isoprostanes. Conclusion: LTE4 induces airflow obstruction and mast cell activation through the CysLT(1) receptor.

Rational: AZD9898 is a small molecule inhibitor of leukotriene C4 synthase (LTC4S) in development for oral treatment of leukotriene driven asthma. AZD9898 is expected to inhibit the downstream production of CysLTs but, unlike the 5-LO inhibitor Zileuton, spare the production of LTB4 and the pro-resolving lipoxins. Hence, AZD9898 has the potential to give additional benefits in asthma compared to leukotriene modifiers on the market. Here we describe the pharmacological properties of AZD9898. Methods: The PK/PD properties and potency of the LTC4S inhibitor AZD9898 was tested in vivo in rat and in vitro in recombinant enzyme assays, human PBMCs, eosinophils and mast cells upon stimulation. Ex vivo stimulation of human whole blood and nasal polyp tissue was used for additional proof of mechanism in human tissue and for investigation of a differentiated profile of AZD9898 compared to CysLT inhibitors currently on the market. Results: The potency of AZD9898 was in the nanomolar range in enzyme assays, rat airways and human PBMCs. AZD9898 completely inhibited LTC4 production in eosinophils and mast cells and showed a differentiated profile compared to current leukotriene modifiers in explant assays of human whole blood and nasal polyps, by inhibiting CysLTs while sparing LTB4 and the pro-resolving lipoxins. There was a direct relationship between plasma concentrations of AZD9898 and CysLT inhibition in the airways of rats. Conclusions: AZD9898 effectively inhibits CysLT production in vivo and in vitro and shows favorable pharmacological properties. LTC4 synthase may be the preferred point at to which inhibit cysteinyl leukotrienes in asthma patients and will be taken forward in clinical studies.

Cysteinyl leukotrienes (cysLTs; LTC 4 , D 4 , E 4 ), are potent biological lipid mediators playing a central pathophysiological role in asthma. Biosynthesis of cysLTs can be induced by the calcium ionophore, A23187. Inhibition of CysLT release in BAL fluid may be used as a target engagement biomarker of a LTC 4 synthase (LTC4s) inhibitor. It is important to characterize the pharmacocinetic-pharmacodynamic (PKPD) relationship of bimoarkers. The aim of this study was to investigate the relationship between plasma concentration and inhibition of CysLT release in lung after single and repeated oral dosing of a LTC4s inhibitor. Sprague Dawley rats were dosed orally with single or multiple doses of a LTC4s inhibitor. Lung lavage with A23187 and blood sampling for determination of the LTC4s inhibitor plasma concentration was performed at different times after dose. Efficacy was measured as % inhibition of CysLTs in BAL fluid compared to controls. Orally administered LTC 4 s inhibitor effectively inhibited CysLT release in BAL fluid in rat following stimulation with calcium ionophore, with a free plasma concentration for 50% effect (EC 50 ) in the same range as in vitro concentration for 50% inhibition (IC 50 ) in human PBMC. The in vivo PKPD relationship was similar irrespective of time after dosing or duration of treatment, suggesting a direct relationship between plasma concentration and inhibition of CysLTs in lung. Our findings support a new opportunity for LTC 4 s inhibition as a therapeutic target for oral treatment of leukotriene-driven asthma. The specific LTC4s inhibitor investigated in our model shows promising properties for clinical use.

Cysteinyl leukotrienes (LTC4, LTD4 and LTE4) are lipid mediators inducing inflammation and bronchoconstriction in allergic asthma and inhibition of cysteinyl leukotriene production is an effective treatment for some asthma patients. Leukotriene A4 (LTA4) is the precursor for both LTC4 and the neutrophil chemoattractant leukotriene B4 (LTB4). The enzyme LTC4 synthase (LTC4s) converts LTA4 into LTC4, while LTA4 hydrolase converts LTA4 into LTB4. A previous study on LTC4s-/- mice showed that loss of LTC4s does not cause shunting towards increased LTB4 production1. If using inhibition of LTC4s as a way to pharmacologically inhibit cysteinyl leukotriene production it is important that no shunting towards LTB4 occurs. In this study we tested whether this lack of shunting holds true also in humans using an in vitro model based on human whole blood. Heparinized whole blood was added to 96 well plate and incubated with different concentrations of an LTC4s inhibitor. Calcium ionophore was added and the plate was incubated for 15 minutes before the reaction was stopped using acetonitrile. LTC4 and LTB4 were measured using mass spectrometry. The production of LTC4 decreased in a dose response manner in response to the LTC4s inhibitor while the LTB4 production was stable. No shunting towards LTB4 production was observed when inhibiting LTC4 production by blocking LTC4s in a human whole blood assay. 1. Kanaoka et al 2001, J Biol. Chem. Vol 276 pp 22608-22613.