Effect of the S-nitrosoglutathione reductase inhibitor N6022 on bronchial hyperreactivity in asthma

Loretta G Que, Zhonghui Yang, Njira L Lugogo, Rohit K Katial, Steven A Shoemaker, Janice M Troha, David M Rodman, Robert M Tighe, Monica Kraft, Loretta G Que, Zhonghui Yang, Njira L Lugogo, Rohit K Katial, Steven A Shoemaker, Janice M Troha, David M Rodman, Robert M Tighe, Monica Kraft

Abstract

Rationale: Patients with asthma demonstrate depletion of the endogenous bronchodilator GSNO and upregulation of GSNOR.

Objectives: An exploratory proof of concept clinical study of N6022 in mild asthma to determine the potential bronchoprotective effects of GSNOR inhibition. Mechanistic studies aimed to provide translational evidence of effect.

Methods: Fourteen mild asthma patients were treated with intravenous N6022 (5 mg) or placebo and observed for 7 days, with repeated assessments of the provocative dose of methacholine causing a 20% fall in FEV1 (methacholine PC20 FEV1), followed by a washout period and crossover treatment and observation. In vitro studies in isolated eosinophils investigated the effect of GSNO and N6022 on apoptosis.

Measurements and main results: This was a negative trial as it failed to reach its primary endpoint, which was change from baseline in methacholine PC20 FEV1 at 24 h. However, our exploratory analysis demonstrated significantly more two dose-doubling increases in PC20 FEV1 for N6022 compared with placebo (21% vs 6%, P < 0.05) over the 7-day observation period. Furthermore, a significant treatment effect was observed in the change in PC20 FEV1 from baseline averaged over the 7-day observation period (mean change: +0.82 mg/ml [N6022] from 1.34 mg/ml [baseline] vs -0.18 mg/ml [placebo] from 1.16 mg/ml [baseline], P = 0.023). N6022 was well tolerated in mild asthmatics. In vitro studies demonstrated enhanced eosinophilic apoptosis with N6022.

Conclusions: In this early phase exploratory proof of concept trial in asthma, N6022 did not significantly alter methacholine PC20 FEV1 at 24 h, but did have a treatment effect at 7 days compared to baseline. Further investigation of the efficacy of S-nitrosoglutathione reductase inhibition in a patient population with eosinophilic asthma is warranted.

Trial registration: ClinicalTrials.gov NCT01316315.

Keywords: N6022; S-nitrosoglutathione; S-nitrosoglutathione reductase; mild asthma.

© 2018 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.

Figures

Figure 1
Figure 1
Study design and patient disposition.
Figure 2
Figure 2
Mean change from baseline in MCh PC20 FEV1 increased over the N6022 post‐treatment observation period compared with the placebo observation period. FEV1, forced expiratory volume in 1 sec; MCh PC20 FEV1, the provocative concentration of methacholine causing a 20% fall in FEV1; SEM, standard error of the mean.
Figure 3
Figure 3
The change from baseline at 24 h in log2‐transformed MCh PC20 FEV1 after N6022 and after placebo for each patient is shown (with the exception of one patient who did not receive placebo). An MCh PC20 FEV1 change of one in log2‐transformed data represents a dose doubling. At 24 h, 5 of 14 patients (36%) had a dose doubling after N6022 and 2 of 13 patients (15%) had a dose doubling after placebo. MCh PC20 FEV1, the provocative concentration of methacholine causing a 20% fall in FEV1.
Figure 4
Figure 4
Percentage of total observations (at 8, 24, and 48 h and Day 7 combined) showing a two dose‐doubling increase in MCh PC20 FEV1 compared with baseline. During the 7‐day post‐treatment observation period, N6022 produced a significant increase in the percentage of observations of two dose‐doubling increases in MCh PC20 FEV1 compared with placebo. FEV1, forced expiratory volume in 1 sec; MCh PC20 FEV1, the provocative concentration of methacholine causing a 20% fall in FEV1.
Figure 5
Figure 5
Investigation of baseline ECP levels and response. (A) Distribution of change from baseline at 24 h in log2‐transformed MCh PC20 FEV1 versus baseline ECP concentration. Each subject's response to N6022 (closed circle) and placebo (open circle) is provided per specific ECP level. For example, the patient with the lowest ECP level of 5 ng/ml (left side of figure) had a log2‐tranformed placebo change from baseline of 0.59 and an N6022 change from baseline of 0.02. The N6022 change from baseline of 2.12 marked with an asterix (*) is the patient with a baseline ECP of 35 ng/ml who withdrew from the study before placebo treatment. Individual patient responses with the same baseline ECP levels to 8 and 10 ng/ml are marked with brackets. An MCh PC20 FEV1 change of one in log2‐transformed data represents a dose doubling. (B) ECP Levels at baseline in responders vs non‐responders in the N6022 treatment period. Responders in this analysis were defined as those patients with a dose‐doubling increase in the MCh PC20 FEV1 compared with baseline at 24 h post‐treatment. ECP, eosinophilic cationic protein; FEV1, forced expiratory volume in 1 sec; MCh PC20 FEV1, the provocative concentration of methacholine causing a 20% fall in FEV1.
Figure 6
Figure 6
Increased eosinophil apoptosis observed in response to N6022 treatment. Flow cytometry analysis of eosinophil 7‐AAD to assess apoptosis in serum eosinophils extracted from IL‐5–transgenic mice. (A) The population with lower forward‐scattered light (FSC) was consistent with apoptotic eosinophils (P1) and the FSC‐high population was consistent with viable eosinophils (P2). (B) N6022 exhibited a dose‐dependent effect on the percentage of 7‐AAD‐positive cells. (C) Western blot analysis of cleaved caspase‐3 to determine apoptosis of serum eosinophils after treatment with N6022 or DMSO vehicle. β‐actin is shown as a loading control. (D) Densitometry performed on the western blot of the control and DMSO‐treated eosinophils vs N6022‐treated eosinophils. Caspase‐3 protein levels were normalized to actin and expressed as arbitrary densitometry units. * P <0.005. 7‐AAD, 7‐amino‐actinomycin D; DMSO, dimethyl sulfoxide; FSC, forward scatter; GSNO, S‐nitrosoglutathione; P, population; SSC, side scatter.

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