Establishment Of Experimental Methodology And Serial Clinical Studies On Leukotriene D4 Bronchial Provocation Test

Rationale and objectives:Bronchial asthma (asthma) is one of the most common chronic respiratory disorders in practice, with the major pathophysiological character of airway inflammation and hyperresponsiveness. Leukotrienes play a pivotal role during the pathogenesis of asthma, as it is capable of eliciting airway spasm, promoting plasma exudation, recruiting inflammatory cells as well as promoting the release of inflammatory mediators. Leukotriene receptor antagonists (LTRA)has gained application in clinics, however, various therapeutic outcomes appear among different asthmatics. It is the very key to predict the actual therapeutic outcome through the measurements available. Due to the vital role of leukotrienes on pathogenesis of asthma as well as its unique impact on severity of airway inflammation, it is reasonable to develop a novel assessment of asthma from another perspective by carrying out leukotriene bronchial provocation test, which would offer an objective proof for proper judgment on the inflammation subtypes (leukotriene sensitive or leukotriene insensitive asthma) as well as disease-oriented application of LTRA. Furthermore, measurement of fractional nitric oxide concentration (FENO) may also help reflect the severity of asthmatic inflammation, but there're few studies available that elucidate the relationship between the test result of FENO and bronchial provocation test (especially for leukotriene bronchial provocation test). The major purpose of this article is to establish the protocol of leukotriene D4 bronchial provocation test for Chinese subjects, investigate its value for diagnosis of asthma and classification of inflammation subtypes, as well as compare the strengths and shortcomings of routine bronchial provocation test (methacholine bronchial provocation test) and measurement of FENO, thereby providing sounding proves for rational selection of measurement and disease-oriented therapies of LTRA.Section 1:Establishment of the methodology of leukotriene D4 bronchial provocation test for Chinese populationBackground: Although leukotriene D4 (LTD4) was proved to be a potent bronchoconstrictor, little is known about the airway responsiveness of LTD4 in asthmatics and normal healthy subjects.Objectives:To establish the methodology and investigate the distribution characters of airway responsiveness, diagnostic value and Adverse events of LTD4 bronchial provocation test.Methods:LTD4 bronchial provocation tests were performed on 62 asthmatics and 21 normal controls. Airway responsiveness was assessed according to the cumulative dose causing a 20% fall in FEV1 (PD20FEV1-LTD4). The fall in spirometric parameters was plotted against logarithmic of cumulative dose. Receiver operation characteristic curve (ROCC) was used to evaluate the diagnostic value. And adverse events were recorded during the test.Results:Airway responsiveness was significantly higher in asthmatics compared to the normal controls (geometric mean:0.530nmol vs 4.370nmol, interquartile range: 0.808nmol vs 0.000nmol). The decrease in spirometric parameters varied after bronchoprovocation, which was negatively correlated with PD20FEV1-LTD4, among which FEV1 had a maximal slope(ρ=-0.524,P=0.000). A high diagnostic value [AUC:0.914, 95%CI:(0.855,0.974)] was revealed by ROCC. The major adverse events were dyspnea (82.3%), chest tightness (72.6%), wheezing (32.3%) and coughing (25.8%) in asthmatics, which could overall be recovered within 5.0 minutes after inhaling 200~400 mcg salbutamol. No serious adverse event was reported.Conclusions:Due to high efficacy and safety, LTD4 bronchial provocation test could be an effective tool for screening asthma and may be useful for identifying anti-leukotriene therapy..Section 2: Airway responsiveness to leukotriene D4 in asthmatics under different control statusBackground: Though leukotriene D4 is capable of eliciting significant bronchoconstriction, the distinction of airway responsiveness in asthmatics under different control status remains unclear.Objectives:To compare the airway responsiveness to leukotriene D4 in asthmatics under different control status.Methods:Leukotriene D4 bronchial provocation tests (LTD4-BPTs) were performed on 20 uncontrolled (UC), 22 partially controlled (PC) and 20 completely controlled (CC) asthmatics and 21 normal controls. The cumulative dose (PD20FEV1-LTD4) was recorded. Comparisons on variation of spirometric parameters, distribution character of PD20FEV1-LTD4, diagnostic parameters and adverse reactions were performed.Results:There was a significant statistical difference (P<0.01) in spirometric parameters except for the change in FVC (t=1.23,P=0.233) after bronchoprovocation. Geometric mean of PD20FEV1-LTD4 was much lower in UC and PC(0.272nmol and 0.386nmol, respectively) compared with CC (1.459nmol). Higher slope (r) of FEV1 fall-log10PD20FEV1 plot was in favor of better asthma control (UC: r= -0.281,P=0.231;PC: r= -0.384,P=0.078;CC: r= -0.721,P=0.000). Sensitivity ranked highest in UC (1.000), followed by PC(0.955) and CC (0.600). Specificity of each group was identical (0.905). Receiver operation characteristic curve (ROCC) revealed the largest area under curve (AUC) in UC (0.998),followed by PC (0.973) and CC (0.721). Fewer adverse events were reported in asthmatics under better control, following the rate of UC> PC> CC.Conclusion:LTD4-BPT offers discriminative power to airway responsiveness in asthmatics under different control status, and it is particularly of diagnostic value to uncontrolled and partially controlled asthmatics.Section 3: Comparison on leukotriene D4 bronchial provocation test and methacholine bronchial provocation test in the diagnosis of asthmaBackground: Despite that leukotriene D4 bronchial provocation test (LTD4-BPT) and methacholine bronchial provocation test (MCh-BPT) are direct measurements of airway responsiveness, their diagnostic value, adverse reactions and capability of screening leukotriene sensitive/insensitive asthmatics remains unclear.Objectives:To compare the difference of both tests and classify leukotriene inflammation subtype in asthmatics.Methods:62 asthmatics, including 20 uncontrolled (UC), 22 partially controlled (PC) and 20 completely controlled (CC) and 21 normal controls were enrolled. All subjects underwent LTD4-BPT and MCh-BPT following the sequence of randomized numbers. Comparisons on distribution character and correlation of cumulative dosages, character of potency ratio, diagnostic value and adverse reactions were performed. Criterion for classifying leukotriene inflammation subtypes was established.Results:The geometric means of PD20FEV1-LTD4 and PD20FEV1-MCh (0.272nmol vs. 0.945μmol) were lowest in UC, followed by PC (0.386nmol vs. 1.933μmol) and CC (1.459nmol vs. 3.946μmol). On molar basis, the average potency ratio was higher in PC (5000.2) than in UC (3477.7) and CC (2702.6). Logarithmic of both cumulative dosages was positively correlated, with the highest correlation (r=0.965, P=0.000) in CC, followed by PC (r=0.661, P=0.001) and UC (r=0.504, P=0.023). Both cumulative dosages were much lower, while potency ratio was much higher in leukotriene-responsive asthmatics compared with leukotriene-unresponsive asthmatics. Receiver operation characteristic curve (ROCC) was slightly in favor of LTD4-BPT [AUC: 0.914, 95%CI: (0.855, 0.974)] over MCh-BPT [AUC: 0.903, 95%CI: (0.839, 0.967)]. The major adverse events were similar, no serious adverse event was reported.Conclusion : Both tests had high diagnostic value with mild adverse events. Leukotriene-responsive/unresponsive asthmatics could be identified through a combination of PD20FEV1-LTD4 and potency ratio.Section 4:Could leukotriene D4 bronchial provocation test be a clear indicator for predicting therapeutic outcomes of leukotriene receptor antagonist?——A pilot studyBackground: Therapeutic outcomes of leukotriene receptor antagonist (LTRA) vary in asthmatics,and there's not an ideal and simple way for prediction at present.Objective:To investigate whether leukotriene D4 bronchial provocation test (LTD4-BPT) could be an indicator of actual therapeutic outcome of LTRA.Methods:A single centre, open-labeled trial was performed in 32 asthmatics with positive LTD4-BPT result for a month. All subjects were categorized according to airway responsiveness to leukotriene D4 (PD20FEV1-LTD4). Subjects received montelukast therapy (10mg, once per night), and reassessment was performed (3~5) days after withholding LTRA. The primary end-points were the difference in pre- and post- treatment PD20FEV1-LTD4.Results:There was no statistical difference (P>0.05) in predicted percentage of FEV1 (FEV1pred%) , airway inflammation (FENO) and airway responsiveness (PD20FEV1-LTD4 and PD20FEV1-MCh) prior to bronchoprovocation in both groups. There was no statistical significance (P>0.05) in PEF measurement in other time points except for the minimal PEF (t=-2.435, P=0.024) and PEFR (Z=-2.002, P=0.045) at week 2. Despite no statistical significance (t=-0.991, P=0.355) in asthma symptom score (AQLQ) in leukotriene-insensitive group, there was a statistical significance (P<0.05) in asthma score in both groups. Logistic regression model suggested no statistical significance in the overall therapeutic benefit (χ~2=11.481, P=0.488). Furthermore, each parameter for assessing the actual therapeutic outcome had a low contribution to the model (P>0.05).Conclusion:The anticipated therapeutic outcome of LTD4-BPT is not correspondent to the actual therapeutic outcome. LTD4-BPT could not be an effective indicator for predicting the therapeutic outcome of LTRA.