Leukotriene E4 induces airflow obstruction and activation through the cysteinyl type 1

Nikolaos Lazarinis, MD,a* Johan Bood, MD, PhD,a,b* Cristina Gomez, PhD,b,c* Johan Kolmert, MSc,b,c* Ann-Sofie Lantz, RN,a*Par€ Gyllfors, MD, PhD,d Andy Davis, PhD,e Craig E. Wheelock, PhD,c* Sven-Erik Dahlen, MD, PhD,b* and Barbro Dahlen, MD, PhDa* Stockholm and Molndal,€ Sweden

GRAPHICAL ABSTRACT

CysLTs cause both bronchoconstricƟon and release of the mast cell mediator PGD2 via the CysLT1 receptor

LTE4

Mast cell acƟvaƟon

CysLT1R ConstricƟon

PGD2 Chemotaxis via CRTH2 ConstricƟon via TP CysLT1R CysLTs: Cysteinyl

PGD2: D2 CysLT1R: Cysteinyl leukotriene type 1 receptor Airway smooth muscle TP: Tromboxane receptor

Background: Leukotriene (LT) E4 is the final active metabolite Objective: We tested this hypothesis by assessing the influence among the cysteinyl leukotrienes (CysLTs). Animal studies have of the CysLT1 antagonist on responses induced by identified a distinct LTE4 receptor, suggesting that current means of inhalation of LTE4 in asthmatic patients. cysteinyl leukotriene type 1 (CysLT1) receptor antagonists can Methods: Fourteen patients with mild intermittent asthma and provide incomplete inhibition of CysLT responses. 2 patients with aspirin-exacerbated respiratory disease received

From athe Division of Respiratory Medicine and Allergy, Department of Medicine, Kar- Disclosure of potential conflict of interest: A. Davis reports employment from olinska University Hospital Huddinge, Stockholm; bthe Unit of Experimental Asthma AstraZeneca AB. S.-E. Dahlen reports personal fees from Merck, GlaxoSmithKline, and Allergy Research, National Institute of Environmental Medicine, and cthe Depart- Regeneron-Sanofi, Teva, and RSPR Pharma and grants and personal fees from ment of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm; dthe AstraZeneca. B. Dahlen reports personal fees from AstraZeneca and Teva. The rest of Stockholm Asthma and Allergy Clinic; and eAstraZeneca Pharmaceuticals, Molndal.€ the authors declare that they have no relevant conflicts of interest. *All authors from Karolinska belong to the Centre for Allergy Research at Karolinska Received for publication September 29, 2017; revised February 16, 2018; accepted for Institutet. publication February 25, 2018. Supported by the Swedish Heart-Lung Foundation, MRC, Stockholm County Council Available online March 5, 2018. Research Funds (ALF), and the Asthma and Allergy Research Foundation. The Corresponding author: Nikolaos Lazarinis, MD, Division of Respiratory Medicine and ChAMP (Centre for Allergy Research Highlights Asthma Markers of Phenotype) Allergy, Department of Medicine, Karolinska University Hospital, Huddinge C2-88, consortium is funded by the Swedish Foundation for Strategic Research, the SE-141 86 Stockholm, Sweden. E-mail: [email protected]. Karolinska Institutet, AstraZeneca & Science for Life Laboratory Joint Research The CrossMark symbol notifies online readers when updates have been made to the Collaboration, and the Vardal Foundation. C.G. and J.K. were supported by article such as errata or minor corrections U-BIOPRED (IMI initiative for severe asthma). C.E.W. was supported by the Swedish 0091-6749/$36.00 Ó Heart-Lung Foundation (HLF 20150640). 2018 American Academy of Allergy, Asthma & Immunology https://doi.org/10.1016/j.jaci.2018.02.024

1080 J ALLERGY CLIN IMMUNOL LAZARINIS ET AL 1081 VOLUME 142, NUMBER 4

20 mg of montelukast twice daily and placebo for 5 to 7 days in a randomized, double-blind, crossover study (NCT01841164). The Abbreviations used PD20 value was determined at the end of each treatment period AERD: Aspirin-exacerbated respiratory disease based on an increasing dose challenge. Measurements included CysLT: Cysteinyl leukotriene CysLT1: Cysteinyl type 1 lipid mediators in urine and sputum cells 4 hours after LTE4 challenge. FENO: Fraction of exhaled nitric oxide IOS: Impulse oscillometry Results: Montelukast completely blocked LTE -induced 4 LT: Leukotriene bronchoconstriction. Despite tolerating an at least 10 times PG: Prostaglandin higher dose of LTE4 after montelukast, there was no difference TX: in the percentage of in sputum. Urinary excretion of all major lipid mediators increased after LTE4 inhalation. Montelukast blocked release of the mast cell product prostaglandin (PG) D2, as well as release of PGF2a and Finally, as an exploratory end point, we collected urine during thromboxane (Tx) A2, but not increased excretion of PGE2 and provocations for analysis of lipid mediator excretion. This led to its metabolites or isoprostanes. the serendipitous discovery of increased urinary excretion of Conclusion: LTE4 induces airflow obstruction and mast cell metabolites of prostaglandin (PG) D2, as well as several other activation through the CysLT1 receptor. (J Allergy Clin potent lipid mediators, after inhalation of LTE . The findings Immunol 2018;142:1080-9.) 4 add a new dimension, namely that LTE4, in addition to having a Key words: Asthma, cysteinyl leukotrienes, receptors, bronchocon- direct bronchoconstrictive action, also activates mast cells and striction, mast cells, sputum cells, leukotrienes, mass spectrometry, other cells to produce secondary responses that can amplify or lipid mediators in urine modify its primary effect.

METHODS The cysteinyl leukotrienes (CysLTs; leukotriene [LT] C4, Fourteen patients with mild intermittent asthma according to Global LTD4, and LTE4) are biosynthesized in mast cells and other in- 8 flammatory cells and contribute to the pathobiology of asthma Initiative for Asthma guidelines and 2 patients with aspirin-exacerbated respiratory disease (AERD) were recruited from our clinic and through by producing bronchoconstriction, increased vascular perme- advertisement. Both patients with AERD had a history of unequivocal severe ability, inflammatory cell infiltration, mucus production, and bronchoconstriction after intake of aspirin-like drugs, and one of them had a 1 smooth muscle proliferation. Although LTC4 and LTD4 cause positive lysine-aspirin inhalation challenge test result.9 Inclusion and bronchoconstriction and many of the proinflammatory effects in exclusion criteria and medications are reported in the Methods section in human subjects through activation of the cysteinyl leukotriene this article’s Online Repository at www.jacionline.org. type 1 (CysLT1) receptor that is blocked by clinically used antag- The Ethical Review Board in Stockholm (Dnr 2011/1016-31/1) and the onists, such as montelukast, the mode of action of LTE4 in asth- Swedish Medical Products Agency (Dnr 151:2011/49661) approved the study. matic patients remains unclear. For example, the relative The participants provided written informed consent. The study is registered at ClinicalTrials.gov (NCT01841164). bronchoconstrictive potency of LTE4 compared with histamine, methacholine, and the other CysLTs has been reported to be greater in asthmatic patients than in healthy subjects,2,3 although Study design 4,5 The study was a double-blind, randomized, placebo-controlled study with a its absolute potency was lower than those of LTC4 and LTD4. Furthermore, studies in genetically modified mice have demon- crossover design involving two 5- to 7-day treatment periods during which the 6 subjects received 20 mg of montelukast twice daily and matching placebo, strated a receptor that is distinctly sensitive to LTE4 and mediates 7 respectively (Fig 1). The dosing and duration of treatment was selected from mucin secretion in the trachea and vascular permeability in the published data on montelukast in LTD challenges.10 6 4 skin. Taken together, the possibility of a distinct E-type receptor At screening, patients’ baseline characteristics and airway sensitivity to in human airways has gained recognition as a potential new target methacholine and inhaled LTE4 were obtained (Table I). A washout period of 7 for the treatment of asthma. However, there are no studies in hu- to 14 days preceded the start of the crossover phase during which inhalation man subjects confirming the presence of such a receptor. challenge with LTE4 was repeated on the last day of each period. Urine samples Therefore in this randomized, placebo-controlled, crossover were collected before, during, and after the end of the challenge at hourly inter- vals for up to 4 hours. For analysis of circulating white blood cells, venous blood study we set out to characterize the effects of inhaled LTE4 in samples were drawn before LTE4 inhalation and at 5 minutes after the last dose. asthmatic patients with a particular focus on whether the CysLT1 receptor was involved. Accordingly, the primary aim of the Sputum induction was performed 4 hours after the end of the LTE4 challenge. investigation was to establish the effect of the intervention with Lung function tests, fraction of exhaled nitric oxide the prototype CysLT1 receptor antagonist montelukast on bronchial responsiveness to increasing dose challenges with measurement, skin prick tests, and sputum inhaled LTE4. The secondary aim of the study was to determine induction the influence of montelukast intervention on airway inflammation Lung function, including spirometry and impulse oscillometry (IOS), measurement of fraction of exhaled nitric oxide (FENO), skin prick tests, and after LTE4 inhalation. This was assessed based on sputum cell counts made after provocations once treatment with placebo or sputum induction were performed as outlined in the Methods section in this article’s Online Repository. active drug had occurred. We hypothesized that if LTE4 was inducing airway inflammation through a different receptor than CysLT1, its effects on sputum cells would be dose dependent Drugs and materials for LTE4 but independent of whether treated with placebo or The CysLT1 receptor antagonist montelukast (Singulair; Merck Sharp & montelukast. Dohme, Stockholm, Sweden) was purchased from the Karolinska University 1082 LAZARINIS ET AL J ALLERGY CLIN IMMUNOL OCTOBER 2018

RESULTS Effects of montelukast on baseline lung function and FENO values There was no difference in prechallenge baseline lung function and FENO values between the 2 treatment periods (see Table E5 in this article’s Online Repository at www.jacionline.org).

Effects of montelukast on airway response to inhaled LTE4 Montelukast protected effectively against LTE4-induced bronchoconstriction: mean maximal FEV1 decrease was 0.45% 2 FIG 1. Study design. *Inhalation challenge with LTE4. Mch, Methacholine; (95% CI, 3% to 2.1%) with montelukast compared with SPT, skin prick test. 26.3% (95% CI, 223% to 229.6%) after placebo (P <.001) and 28.7% (95% CI, 225.3% to 232%) at screening (Fig 2, Hospital Pharmacy, and matching placebo was obtained from Recipharm A). After treatment with montelukast, all subjects tolerated the Pharmaceutical Development AB (Stockholm, Sweden). High-purity LTE4 for human use was purchased from Cayman Chemicals (Ann Arbor, Mich). highest cumulated dose of inhaled LTE4 (geometric mean, 380,277 pmol), which was, on average, 10-fold higher compared For safety reasons, LTE4 solutions were dispersed in color-coded vials in 10-fold increasing concentrations from 0.042 to 4200 mmol/L (see the with placebo (geometric mean, 37,006 pmol; P <.001;Fig 2, B). Methods section in this article’s Online Repository). There was no significant difference in LTE4 PD20 between screening and placebo treatment (geometric mean, 23,610 vs Bronchoprovocation 20,923 pmol; Fig 2, B); likewise, Bland-Altman analysis LTE4 was administered for inhalation after nebulization by using a dosimeter- indicated excellent repeatability for the challenge (see Fig E8 controlled jet nebulizer (Spira Elektro 2; Respiratory Care Center, Hameenlinna, in this article’s Online Repository at www.jacionline. Finland), with pulmonary function measured as FEV . IOS measurements were 1 org). Compared with methacholine, LTE4 was about 77 times done before and 5 minutes after the end of the challenge. Challenges were per- more potent on a molar basis (Table I). Furthermore, LTE4 was formed in the morning and started by inhalation of the diluent. Provided FEV1 a relatively more potent bronchoconstrictor in the subjects was stable (ie, not deviating >10%), the postdiluent FEV value was used as a 1 with the lowest airway hyperresponsiveness to methacholine baseline value. Half-log dose increments of LTE solution were inhaled every 4 (Fig 2, C). 10 minutes, and FEV1 was obtained after each dose increment. Provocation IOS measurements were performed in 11 subjects, including was terminated when FEV1 had decreased by 20% from the baseline value or the maximum dose of LTE4 was reached. The PD20 value was derived by means both patients with AERD, to extend the information about the of linear interpolation from the log-cumulated dose-response curve. Bronchial influence of LTE4 on the airway tree. There was a mean 58.9% hyperresponsiveness to methacholine was assessed with a similar protocol but change (95% CI, 41.1% to 76.8%) in total airway resistance at with dose increments every third minute in doubling doses (88.6- 5 Hz after inhalation of LTE4 with placebo compared with a 45,274 nmol). For the 2 patients with AERD, asthma medications were withheld mean 7.1% change (95% CI, 23.2% to 17.2%) with montelukast 24 hours before provocation. The protocols for the provocations are in Tables E3 (P < .001). With respect to the sensitive markers of small-airway and E4 in this article’s Online Repository at www.jacionline.org. obstruction,14 frequency-dependent resistance and reactance area, there were 7- and 10-fold increases, respectively, after inha- Measurement of urinary metabolites lation of LTE4 in the presence of placebo. This conspicuous Urine was stored at 2708C until assay. COX metabolites of arachidonic small-airway constriction caused by LTE4 was abolished by mon- A B acid, LTE4, and isoprostanes were measured by using in-house ultraperform- telukast (Fig 3, and ). Changes in all measured IOS parameters ance liquid chromatography/tandem mass spectrometry method.11 Mass spec- during the 2 treatment periods are presented in Table E6 in this trometry measurements of PGD2 metabolites were compared with the article’s Online Repository at www.jacionline.org. 12 immunoassay for 11b-PGF2a (Cayman Chemical), as described previously. Effects of montelukast on counts Urinary samples from the validation cohort Five minutes after inhalation of the last dose of LTE4, there was Urinary samples collected during a previous bronchoprovocation study an increase in numbers of circulating white blood cells, which 13 with LTD4 in healthy subjects and patients with asthma were analyzed. Sam- was driven by increased numbers of total lymphocytes and asso- ples had been biobanked at 2208C for 12 years. ciated with a distinct decrease in numbers of eosinophils, whereas and monocyte numbers remained the same (see Fig E1 in this article’s Online Repository at www.jacionline.org). How- Statistical analysis ever, there was no significant difference between placebo and Statistical analyses were performed with GraphPad Prism 6.0c software montelukast with respect to these changes in white blood cell (GraphPad Software, La Jolla, Calif). PD20 FEV1 values for LTE4 and FENO values were transformed logarithmically, data were presented as geometric counts (see Fig E1). means and ranges, and repeated-measures ANOVA was performed. Correla- tions were performed with Pearson correlation on log-transformed data. Because of nonnormal distribution, data from IOS measurements and total Effects of montelukast on sputum cells sputum cell counts were presented as medians with ranges, and the Wilcoxon Paired data on viable sputum cells were obtained in 9 matched-pairs signed rank test was used for analysis. Paired t tests were used subjects (including 1 patient with AERD) 4 hours after LTE4 to determine changes in percentages of blood differential cell counts, as well challenge on the last day of each treatment period. There as changes in levels of urinary metabolites. was no significant difference between placebo and montelukast J ALLERGY CLIN IMMUNOL LAZARINIS ET AL 1083 VOLUME 142, NUMBER 4

TABLE I. Patient characteristics at screening

Screening LTE4 PD20 MCh PD20/LTE4 PD20 Patient ID Age (y) M/F FENO (ppb) FEV1 (L) FEV1 (% predicted) MCh PD20 (nmol) (nmol) ratio 1 45 F 8 2.1 79 4,986 3.8 1,326 2 24 M 68 4.2 86 1,654 54.9 30.2 3 37 F 50 2.9 114 499 39.4 12.7 4 28 F 13 2.8 81 2,565 30.8 83.4 5 38 M 116 4.3 106 916 70.2 13 6 29 M 24 4.3 90 2,378 19.8 120.6 7 46 F 10 2.7 99 3,532 49.4 71.5 8 20 F 11 3.5 95 2,153 31.9 67.4 9 52 F 14 2.6 101 12,188 43.2 282 10 45 F 36 2.4 96 924 8.6 107.3 11 25 F 30 2.9 92 899 11.3 79.5 12 44 F 52 4.1 129 4,281 22.7 188.4 13 38 F 11 2.5 103 2,197 12.6 173.8 14 28 F 48 2.6 93 649 2.9 224 AERD 1 48 M 14 3.8 97 17,206 87.4 197 AERD 2 35 M 21 3.8 79 2,528 55.7 45.4 Mean 36.4 24.2* 3.2 96.3 1,815* 23.6* 76.9 Range 20-52 8-116 2.1-4.3 79-129 499-17,206 2.9-54.9 12.7-1,326

F, Female; M, male; MCh, methacholine. *Geometric mean.

* * A 20 B 1 106

0 100000 change % pmol 1

-20 4 10000 LTE -40 1000 Max FEV

-60 100 Screening Placebo Montelukast Screening Placebo Montelukast

C

(nmol) 4 20 PD 4 3

2 - log LTE 20

1

log Mch PD 0 2.5 3.0 3.5 4.0 4.5

log Mch PD20(nmol)

FIG 2. A, Maximal FEV1 change percentage at screening (blue plot) and after treatment with placebo (red plot) and montelukast (green plot). Open circles represent values for patients with AERD. Horizontal bars

indicate mean values. *P < .001 between treatments. B, LTE4 PD20 values at screening (blue plot) and after treatment with placebo (red plot) and total dose of inhaled LTE4 after treatment with montelukast (green plot). Open circles represent values for patients with AERD. Horizontal bars indicate geometric mean values.

*P < .001 between treatments. C, Relation between airway responsiveness to methacholine (PD20) and rela- tive potency of LTE4 compared with methacholine (Mch; difference in logPD20 methacholine and logLTE4 PD20 values). r 5 0.57, P 5 .002 (Pearson). with respect to the percentage of eosinophils (mean, 4.5% (median, 4.27 [interquartile range, 2.4-8.8 3 106/g sputum] vs [95% CI, 1.8% to 7.1%] for placebo vs 3.2% [95% CI, 1.4% 5.3 [interquartile range, 3.6-9.9 3 106/g sputum] for placebo to 4.9%] for montelukast; P 5 .11; Fig 3, C)orneutrophils and montelukast, respectively; P 5 .039; Fig 3, D). Other (mean, 28.8% [95% CI, 18.5% to 39%] for placebo vs differential cell counts were also similar between the 2 30.4% [95% CI, 12.9% to 47.9%] for montelukast; P 5 .82), treatment periods (see Fig E2 in this article’s Online although there was a small increase in the total number of cells Repository at www.jacionline.org). 1084 LAZARINIS ET AL J ALLERGY CLIN IMMUNOL OCTOBER 2018

AB0.4 * * 6 0.3 *

0.2 4 * 0.145 0.1 1.62 Ax (kPa/L) 2 0.02 R5-R20 (kPa/L/s) 0.02 0.01 0.0 0.15 0.16 -0.1 0 0.14 Pre Post Pre Post Pre Post Pre Post

Placebo Montelukast Placebo Montelukast CD 15 * 25 /g) 6 12 20 9 15

6 10 4.5 Sputum Eos % 3.2 5.3 3 5 4.3

0 sputum cell count (x10 Total 0 Placebo Montelukast Placebo Montelukast

FIG 3. A and B, Changes in frequency-dependent resistance (R5–R20; Fig 3, A) and reactance area (Ax; Fig 3, B) before and 5 minutes after LTE4 inhalation challenge for both treatment periods. Horizontal bars indicate median values. *P < .01, Wilcoxon signed rank test. C and D, Effects of montelukast on sputum (Eos; Fig 3, C) and total sputum cell (Fig 3, D) counts from 9 subjects with paired data. The open circle rep- resents values in patient number 2 with AERD. Horizontal bars indicate median (Fig 3, C) and mean (Fig 3, D) values. *P 5 .039.

ABLTE4 5

* ** 9000 4 8000 -1 7000 3 6000 ** 5000 2

4000 (ng) urine excretion 4 3000 2000 1 ng.mmol creatinine 1000 log LTE 0 0 Base Peak Base Peak 3.0 3.5 4.0 4.5 5.0 5.5 6.0

Placebo Montelukast log LTE4 inhaled cumulative dose (ng)

FIG 4. A, Urinary excretion of LTE4 before and after challenge with inhaled LTE4 during the 2 treatment pe- riods. Horizontal bars indicate mean values, with upper bars for before versus after values during each period and lower bars for comparison between periods. Measurements were performed with ultraperform- ance liquid chromatography/tandem mass spectrometry, and values are expressed as nanograms per milli-

mole of creatinine *P < .01 and **P < .001. B, Relation between inhaled and excreted LTE4 (both expressed as nanograms) in the urine for both treatment periods (r 5 0.68, P < .0001 [Pearson]).

Mediator excretion in urine (all data are presented baseline, P 5.0002). Peak excretion during montelukast treatment as nanograms per nanomole of creatinine) was significantly greater (P 5 .002; Fig 4, A). There was a consis- LTE . As expected, urinary excretion of LTE increased tent 2% recovery of inhaled LTE4 (1.7% on placebo and 2.4% on 4 4 B compared with baseline values after inhalation of LTE4 during montelukast) across the range of inhaled concentrations (Fig 4, ). both treatment periods (981 6 240 after placebo vs 15 6 10 at PGD2 metabolites. Levels of the most abundant metabolite, baseline, P 5 .0015; 3201 6 645 after montelukast vs 11 6 5at tetranor-PGDM, increased after LTE4 inhalation on placebo J ALLERGY CLIN IMMUNOL LAZARINIS ET AL 1085 VOLUME 142, NUMBER 4

Tetranor-PGDM AB2,3DN-11 -PGF2a * 18000 10000 -1 -1 16000 14000 12000 5000 8000 6000 2000 4000 ng.mmol creatinine

ng.mmol creatinine 1000 2000 0 0 Base Peak Base Peak Base Peak Base Peak

Placebo Montelukast Placebo Montelukast

11 -PGF2a EIA C * 1500

-1 1000 500 * 400 300 200 ng.mmol creatinine 100 0 Base Peak Base Peak

Placebo Montelukast

FIG 5. Urinary excretion of the major PGD2 metabolites, as measured by using ultraperformance liquid chromatography/tandem mass spectrometry (*P < .05; A and B) and an EIA (C). Horizontal bars indicate mean values. *P < .05. All values are expressed as nanograms per millimole of creatinine.

(3743 6 1335 vs 272 6 77 at baseline, P 5.0189). This increase are presented in the Results section and Fig E4 in this article’s On- was completely blocked during montelukast treatment line Repository at www.jacionline.org. (509 6 101 vs 418 6 135 at baseline, not significant; Fig 5, A). PGF2a. PGF2a levels increased significantly after challenge The early metabolite of the mast cell mediator PGD2, 2,3-dinor- during placebo treatment (711 6 158 vs 209 6 34 at baseline, 11b-PGF2a, showed an identical pattern (Fig 5, B), although the P 5 .0029) but did not reach a significant increase during numeric mean increase during placebo did not reach statistical montelukast treatment (411 6 67 vs 251 6 74 at baseline, significance (P 5 .072). P 5 .06). Peak excretion was greater during placebo treatment We replicated the data for urinary excretion of PGD2 metab- compared with that during the montelukast period (P 5 .0350; olites observed in the ultraperformance liquid chromatog- Fig 6, B). raphy/tandem mass spectrometry analysis by using a Prostacyclin metabolite 2,3-dinor-6-keto-PGF1a. This commercially available EIA for the very early PGD2 metabo- metabolite did not increase significantly after LTE4 inhalation lite 11b-PGF2a (Fig 5, C). However, this metabolite was not with either treatment. However, there was a trend (P 5 .055) to- detected by using mass spectrometry, extending earlier data ward an increased level during placebo treatment (Fig 6, C). indicating that the 10% cross-reactivity of the antibody against PGE2 and its metabolites. Urinary excretion of primary 11b-PGF2a with 2,3-dinor-11b-PGF2a explains the results of PGE2 increased to the same extent after challenge during both using that particular EIA on urine.12,15 We confirmed good treatment periods (143 6 31 for placebo vs 57 6 21 at baseline, agreement between the 2 measurements by using Bland- P 5 .0219; 147 6 34 for montelukast vs 69 6 39 at baseline, Altman analysis (see Fig E3 in this article’s Online Repository P 5 .0188; Fig 6, D). The same was true for the later and more at www.jacionline.org). abundant metabolite tetranor-PGEM (1555 6 365 for placebo Thromboxane B2 and its metabolites. Levels of the vs 363 6 111 at baseline, P 5.0056; 1823 6 407 for montelukast most abundant metabolite, 2,3DN-TXB2, increased after LTE4 vs 388 6 90 at baseline, P 5 .0037; Fig 6, E). inhalation challenge during both treatments (1853 6 460 for pla- Isoprostanes. Levels of the most abundant metabolite, 8,12- cebo vs 201 6 74 at baseline, P 5 .0019; 561 6 116 for monte- iPF2a-VI, increased after LTE4 inhalation challenge during both lukast vs 159 6 41 at baseline, P 5 .0021). Peak excretion was treatments (2139 6 347 for placebo vs 858 6 166 at baseline, significantly greater with placebo (P 5.0092; Fig 6, A). Measure- P 5 .0035; 1756 6 273 for montelukast vs 1081 6 220 at ments of primary thromboxane (TX) B2 and 11DH-TXB2 levels baseline, P 5 .0274; Fig 6, F). Results of 8iso-PGF2a and 1086 LAZARINIS ET AL J ALLERGY CLIN IMMUNOL OCTOBER 2018

2,3DN-TXB2 2,3DN-6K-PGF ABPGF2a C1a ** ** ** 6000 3000 20000

-1 1

5000 - -1 e 15000 2000 4000 ** * 3000 10000

2000 1000 mmol creatinin mmol 5000

ng.mmol creatinine ng. ng.mmol creatinine 1000

0 0 0 Base Peak Base Peak Base Peak Base Peak Base Peak Base Peak

Placebo Montelukast Placebo Montelukast Placebo Montelukast D Tetranor-PGEM F 8,12-iPF2a-VI PGE2 E ** ** ** * * 7000 5000 1000 * 6000 -1 -1 4000

-1 800 5000 600 4000 3000 3000 400 2000 2000 ng.mmol creatinine ng.mmol creatinine ng.mmol creatinine 200 1000 1000 0 0 0 Base Peak Base Peak Base Peak Base Peak Base Peak Base Peak Placebo Montelukast Placebo Montelukast Placebo Montelukast

FIG 6. Urinary excretion of other important and their major metabolites measured with ultraperformance liquid chromatography/tandem mass spectrometry during the 2 treatment periods.

(A) 2,3DN-TXB2, (B) PGF2a, (C) 2,3DN-6K-PGF1a, (D) PGE2, (E) Tetranor-PGEM, (F) 8,12-iPF2a-VI. All values are expressed as nanograms per millimole of creatinine. Horizontal bars indicate mean values, with upper bars for before versus after differences during each period and lower bars displaying significant differences be- tween periods. *P < .05 and **P < .01.

AB2,3DN-PGF2 LTE4 * 1000 ** -1 * -1 ** 1000 *** * ** * 800

600

500 400

200 ng.mmol creatinine ng.mmol creatinine 0 0

Base Peak base Base Peak Peak Base Peak base Peak Base Peak

Healthy Asthma Healthy Asthma asthma +ICS asthma +ICS

FIG 7. Urinary excretion of the PGD2 metabolite 2,3-dinor-PGF2a (A) and LTE4 (B) after LTD4 inhalation chal- lenge, as measured with ultraperformance liquid chromatography/tandem mass spectrometry. All values are expressed as nanograms per millimole of creatinine. *P < .05, **P < .01, and ***P < .001. ICS, Inhaled corticosteroid.

2,3DN-8iso-PGF2a measurement are presented in the Results sec- 2,3DN-b8iso-PGF2a, and 8,12-iPF2a;LTE4. Data for 2,3DN- tion and Fig E5 in this article’s Online Repository. PGF2a and LTE4 are shown in Fig 7, and data for the other metabolites are reported in Fig E6 in this article’s Online Repository at www.jacionline.org. No significant increases were Validation of urinary mediator excretion in seen for the following detected mediators: primary TXB2 biobanked samples from bronchoprovocation with and 11DH-TXB2, PGF2a, and the prostacyclin metabolite LTD4 2,3DN-6K-PGF1a (see Fig E7 in this article’s Online Repository Analysis of urine collected in a previous LTD4 bronchoprovo- at www.jacionline.org). We were unable to detect measurable 13 cation study found increased urinary excretion of the following amounts of primary PGE2, tetranor-PGEM, or tetranor-PGDM, compounds after LTD4: the PGD2 metabolite 2,3DN-PGF2a; the which is in line with previous studies reporting on their 16 TXB2 metabolite 2,3-DN-TXB2; the isoprostanes 8iso-PGF2a, degradation during storage. J ALLERGY CLIN IMMUNOL LAZARINIS ET AL 1087 VOLUME 142, NUMBER 4

DISCUSSION endogenous CysLTs contribute to small-airway disease. The In this controlled study we demonstrate that treatment for 5 to small-airway obstruction induced by LTE4 might be due to both 7 days with the selective CysLT1 receptor antagonist montelukast smooth muscle constriction and mucosal edema because CysLTs 25 effectively inhibited a broad range of responses induced by LTE4 are potent inducers of plasma exudation, which is also shown in inhalation. This is the first study in which montelukast, the clini- the airways.26,27 cally most widely used CysLT1 antagonist, has been assessed for We deliberately used a higher dose of montelukast (40 mg) than its efficacy against airway obstruction induced by LTE4. In addi- the ordinary clinical 10 mg once-daily dosing. This was because tion, we discovered that inhaled LTE4 led to increased urinary we wished to ensure effective CysLT1 antagonism to avoid ambig- levels of several major metabolites of -derived lipid uous data caused by incomplete receptor inhibition by this mediators; in particular, increased excretion of metabolites of competitive antagonist. In fact, the only published dose-ranging PGD2 suggests that the CysLT1 receptor in vivo can activate study of montelukast in the LTD4 provocation setting has shown mast cells. Furthermore, LTE4-induced release of PGD2 metabo- progressively increasing shifts in the dose-response relation for lites was blocked by montelukast and replicated by inhalation of LTD4 up to 250 mg of montelukast. It should be appreciated LTD4, which together lend strong support to the interpretation that montelukast is a very selective CysLT1 antagonist and that that this presumed mast cell activation was mediated by the the initial clinical treatment studies used much higher doses 28,29 CysLT1 receptor. In fact, this new finding heralds a new awareness (100-200 mg). Interestingly, lung function improvement in that CysLTs should be reclassified as both directly and indirectly these high-dose studies was greater than in subsequent studies us- acting bronchoconstrictors. ing 10 mg,30 although performed on comparable patient popula- As expected, this study confirmed that LTE4 is a more potent tions. Perhaps it is time to revisit the dosing of montelukast? bronchoconstrictor than methacholine. Although the bronchocon- Furthermore, despite exposure to one order of magnitude strictive properties of LTD4 have been studied extensively, there higher dose of LTE4 in the presence of montelukast, the sputum are only a handful studies of how the terminal CysLT LTE4 affects eosinophil count was the same as during the placebo period. If human airways in vivo. Compared with methacholine, LTE4 was another receptor had been sensitive to LTE4, the substantially about 70 times more potent on a molar basis. Using an identical higher agonist concentration would have been expected to result challenge protocol, we found that inhaled LTD4 is approximately in an amplified response. Therefore the finding argues against 1000 times more potent than methacholine.13 This confirms the additional receptors being involved in the proinflammatory ef- potency differences determined in the few studies in which fects of LTE4 on airway cells. Inhaled LTD4 and LTE4 levels inhaled LTE4 has been compared with methacholine or histamine have been reported to differ with respect to the ability to cause 3 31 on the one hand and LTC4 or LTD4 on the other hand. We also airway eosinophilia. Our study suggests that the outstanding found that the asthmatic patients with the highest hyperrespon- explanation for this observation is unlikely to be related to a siveness for methacholine had the lowest relative airway respon- different receptor for LTE4. siveness to LTE4, which is in perfect agreement with relations Original work on LT mechanisms in animal airways showed that established between methacholine and inhaled LTC4 or LTD4 CysLTscan cause profound release of PGs and other COX products levels in previous studies.13,17 that contribute to the overall biological responses32,33; however, Furthermore, pretreatment with montelukast abolished bron- this mechanism has never been confirmed in human subjects in vivo choconstriction after inhalation of LTE4 in all study subjects (14 . For the first time, our study demonstrated that inhaled patients with mild asthma and 2 patients with AERD). During LTE4 induces increased urinary excretion of metabolites indicative treatment with montelukast, on average, asthmatic patients toler- of pulmonary release of PGE2,PGD2,PGF2a, and TXA2,aswellas 34 ated a 10-fold higher dose of inhaled LTE4 compared with pla- several isoprostanes being markers of oxidative stress. cebo without any decrease in lung function. In contrast, during Increased excretion of PGD2 is considered a sign of mast cell the placebo-treated period, the subjects showed an excellent activation because this cell is the major source of PGD2 in human 12,35 repeatability of LTE4 PD20 measurements determined during subjects. This increased excretion was abolished by montelu- screening. Our findings are in agreement with those of previous kast treatment, confirming it to be CysLT1 dependent. There are studies using 2 other LT receptor antagonists against inhaled previous experimental data supporting that CysLTs can activate 5,18 36,37 LTE4. The results support that the in vivo effect of LTE4 on mast cells and cause secondary release of . More- airway smooth muscle in human subjects is mediated exclusively over, we were able to replicate the effect of LTE4 on PGD2 release by the CysLT1 receptor. This agrees with in vitro data in isolated by measurements in urine that had been biobanked after a previous 13 human bronchi and other models in which LTE4 acts through the LTD4 inhalation study. That study also supported dose- 1,19-21 CysLT1 receptor. dependent mast cell activation because it included healthy subjects Because FEV1 reflects mainly changes to the larger airways, who tolerated a higher dose of LTD4 and displayed a more pro- for the first time, this study applied IOS measurements in subjects nounced mediator excretion in urine. Because mast cells also pro- challenged with LTE4. This approach revealed pronounced pe- duce CysLTs, the study results raise the hypothesis that CysLT1- ripheral airway obstruction after LTE4 inhalation in the presence dependent mast cell activation might represent a positive feedback of placebo similar to that seen in patients with severe asthma and loop sustaining the response to the initial stimulus. Another impli- small-airways disease.22 In contrast, during treatment with mon- cation of this new finding is that eosinophils, which are proficient telukast, there were no signs of small-airways impairment and producers of CysLTs, could prime mast cells by this particular essentially no significant changes in IOS parameters after LTE4 mechanism. The allegation of a prostanoid component in LT- inhalation. Interestingly, previous treatment studies with monte- induced airway hyperresponsiveness was previously indicated by lukast have indicated that small-airway function in asthmatic pa- the finding of Christie et al38 that indomethacin blocked the in- 23,24 tients was improved by montelukast, implicating that crease in histamine responsiveness that followed LTE4 inhalation. 1088 LAZARINIS ET AL J ALLERGY CLIN IMMUNOL OCTOBER 2018

TXA2 release and, to some extent, also PGF2a showed similar 7. Bankova LG, Lai J, Yoshimoto E, Boyce JA, Austen KF, Kanaoka Y, et al. Leuko- sensitivity to inhibition by montelukast. This could be also be due triene E4 elicits respiratory epithelial cell mucin release through the G-protein- coupled receptor, GPR99. Proc Natl Acad Sci U S A 2016;113:6242-7. to CysLT1-mediated release from mast cells, but secondary 8. From the Global Strategy for Asthma Management and Prevention, Global Initia- release from other sources is also possible. It remains poorly char- tive for Asthma (GINA) 2015. Available at: http://www.ginasthma.org. Accessed acterized which lipid mediators are formed in human mast cells May 20, 2015. 9. Nizankowska-Mogilnicka E, Bochenek G, Mastalerz L, Swierczynska M, Picado in vivo. In contrast, increased urinary excretion of PGE2 and its major metabolite, tetranor-PGEM, were not blocked by montelu- C, Scadding G, et al. EAACI/GA2LEN guideline: aspirin provocation tests for diagnosis of aspirin hypersensitivity. Allergy 2007;62:1111-8. kast. It is possible that another receptor might be involved in that 10. De Lepeleire I, Reiss TF, Rochette F, Botto A, Zhang J, Kundu S, et al. Montelu- release, perhaps from the airway epithelium, which is a major kast causes prolonged, potent leukotriene D4-receptor antagonism in the airways of 39,40 source of PGE2. However, because the patients received a patients with asthma. Clin Pharmacol Ther 1997;61:83-92. 11. Balgoma D, Larsson J, Rokach J, Lawson JA, Daham K, Dahlen B, et al. Quanti- 10-fold greater dose of LTE4 in the presence of montelukast fication of lipid mediator metabolites in human urine from asthma patients by elec- without an increase in urinary excretion of PGE2, it is also trospray ionization mass spectrometry: controlling matrix effects. Anal Chem possible that the effect partly involved the CysLT1 receptor. 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