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 PC20FEV1 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.
This is an early stage proof of concept cross‐over trial for an S‐nitrosoglutathione reductase inhibitor, N6022, in 14 patients with mild non‐corticosteroid treated asthma. Although the study did not reach its primary endpoint of a reduction in methacholine PC 20 FEV1 at 24 h, a reduced methacholine PC 20 FEV1 at 7 days compared with baseline was achieved.
The importance of nitric oxide (NO) in hypoxic pulmonary hypertension has been demonstrated using nitric oxide synthase (NOS) knockout mice. In that model NO from endothelial NOS (eNOS) plays a ...central role in modulating pulmonary vascular tone and attenuating hypoxic pulmonary hypertension. However, the normal regulation of NOS expression in mice following hypoxia is uncertain. Because genetically engineered mice are often utilized in studies of NO, we conducted the present study to determine how hypoxia alters NOS expression in wild-type mice.
Mice were exposed to sea level, ambient conditions (5280 feet) or severe altitude (17,000 feet) for 6 weeks from birth, and hemodynamics and lung NOS expression were assessed.
Hypoxic mice developed severe pulmonary hypertension (right ventricular systolic pressure RVsP 60 mmHg) as compared with normoxic mice (27 mmHg). Using quantitative reverse-transcription PCR, it was found that expressions of eNOS and inducible NOS (iNOS) increased 1.5-fold and 3.5-fold, respectively, in the lung. In addition, the level of lung eNOS protein was increased, neuronal NOS (nNOS) protein was unchanged, and iNOS was below the limit of detection. Immunohistochemistry demonstrated no change in lung iNOS or nNOS staining in either central or peripheral areas, but suggested increased eNOS in the periphery following hypoxia.
In mice, hypoxia is associated with increases in lung eNOS, possibly in iNOS, but not in nNOS; this suggests that the pattern of lung NOS expression following hypoxia must be considered in studies using genetically engineered mice.
Nitric oxide plays an important role in modulating pulmonary vascular tone. All three isoforms of nitric oxide synthase (NOS), neuronal (nNOS, NOS I), inducible (iNOS, NOS II), and endothelial (eNOS, ...NOS III), are expressed in the lung. Recent reports have suggested an important role for eNOS in the modulation of pulmonary vascular tone chronically; however, the relative contribution of the three isoforms to acute modulation of pulmonary vascular tone is uncertain. We therefore tested the effect of targeted disruption of each isoform on pulmonary vascular reactivity in transgenic mice. Isolated perfused mouse lungs were used to evaluate the effect of selective loss of pulmonary nNOS, iNOS, and eNOS with respect to hypoxic pulmonary vasoconstriction (HPV) and endothelium-dependent and -independent vasodilation. eNOS null mice had augmented HPV (225 +/- 65% control, P < 0.02, mean +/- SE) and absent endothelium-dependent vasodilation, whereas endothelium-independent vasodilation was preserved. HPV was minimally elevated in iNOS null mice and normal in nNOS null mice. Both nNOS and iNOS null mice had normal endothelium-dependent vasodilation. In wild-type lungs, nonselective NOS inhibition doubled HPV, whereas selective iNOS inhibition had no detectable effect. In intact, lightly sedated mice, right ventricular systolic pressure was elevated in eNOS-deficient (42.3 +/- 1.2 mmHg, P < 0.001) and, to a lesser extent, in iNOS-deficient (37.2 +/- 0.8 mmHg, P < 0.001) mice, whereas it was normal in nNOS-deficient mice (30.9 +/- 0.7 mmHg, P = not significant) compared with wild-type controls (31.3 +/- 0.7 mmHg). We conclude that in the normal murine pulmonary circulation 1) nNOS does not modulate tone, 2) eNOS-derived nitric oxide is the principle mediator of endothelium-dependent vasodilation in the pulmonary circulation, and 3) both eNOS and iNOS play a role in modulating basal tone chronically.
This review highlights the phenotypic features that lead to the diagnosis of cystic fibrosis in adults, and the prognosis of these patients.
With the widespread availability of genetic testing and a ...greater appreciation of the clinical spectrum of the disease, the diagnosis of cystic fibrosis is being made with increasing frequency in adults. Clinical features that lead to the diagnosis include respiratory symptoms and chronic airway infection with typical cystic fibrosis pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus, as well as nontuberculous mycobacteria. Often these patients have previously received diagnoses of asthma, chronic bronchitis, or emphysema. Pancreatic insufficiency is much less common in the adult receiving the diagnosis, but pancreatitis occurs with greater frequency. Occasionally, individuals receive diagnoses of apparent single-organ manifestations such as idiopathic pancreatitis or congenital bilateral absence of the vas deferens, but with negligible involvement of the respiratory tract. On rare occasions, patients receiving the diagnosis as adults can present with classic features of the disease. Although lung disease is generally less severe in cystic fibrosis patients receiving the diagnosis as adults than in adult patients who received the diagnosis as infants, the extent of bronchiectasis can nonetheless be severe. The clinical course of patients receiving a diagnosis of cystic fibrosis in adulthood is largely unknown, but frequently they have milder disease and a more favorable prognosis.
Clinicians must be aware of the potential for adults with chronic respiratory tract infections, unexplained bronchiectasis, congenital bilateral absence of the vas deferens, or pancreatitis to have cystic fibrosis despite the age at presentation.
The United States displayed a keen interest in the nature, progress and results of the Yom Kippur War, because the fighting was thought to reflect how non-nuclear hostilities between the North ...Atlantic Treaty Organization (NATO) and the Warsaw Pact (WP) would unfold on the plains of central Europe in case war broke out there. In contrast to many observers of the war, who concluded that the losses suffered by the Israel Defence Forces (IDF) at the hands of Egyptian and Syrian anti-aircraft and anti-tank weapons had rendered the aircraft and the tank largely impotent, thereby revolutionizing how wars would be waged in the future, Central Intelligence Agency (CIA) analysts and United States Army Training and Doctrine (TRADOC) Command officers, based upon an in-depth review of the nature, progress and results of the fighting, re-affirmed the centrality of these weapons systems on the modern battlefield. Rather than focus obsessively on technological developments, they concluded that training, leadership and tactics were ultimately the decisive elements in the Yom Kippur War.
Gene therapy for pulmonary disease has attracted a great deal ofattention since the first report of successful gene delivery 10 yearsago. Potential indications for gene therapy include chronic ...illnessessuch as cystic fibrosis and α1-antitrypsin deficiency, and acute illnesses such as acute transplant rejection andchemotherapy-induced lung injury. The key technological impediment tosuccessful gene therapy is vector optimization. Viral vectors, including adenovirus and adeno-associated virus, have relatively lowefficiency in vivo. In addition, adenovirus has beenassociated with a brisk inflammatory response and limited duration ofexpression in the lung. Nonviral vectors, particularly liposomes, havealso been tried, with limited expression efficiency and some toxicity. Although work is ongoing to improve adenoviral and adeno-associatedviral vectors and test other viral and nonviral vectors, an idealvector has not yet been identified. Several important barriers tosuccessful gene therapy, including the host inflammatory response, promotor down-regulation, tissue-specific targeting, and physicalbarriers to gene delivery in the airway, will need to be overcome. Despite these daunting problems, several human gene therapy trials havebeen completed, using adenovirus, adeno-associated virus, andliposomes. In general, these trials have been focused on safety, andhave shown that there is dose-dependent inflammation in response toadenovirus. Adeno-associated virus appears to cause littleinflammation. Demonstration of successful gene delivery andtranscription has been quite variable in human trials. In general, thelevel of expression of transgene appears to be quite low. In summary, although there is great promise for gene therapy in the lung, significant challenges remain in translating this technology tosuccessful human therapy.
Circulating leukocyte RNA transcripts are systemic markers of inflammation, which have not been studied in cystic fibrosis (CF) lung disease. Although the standard assessment of pulmonary treatment ...response is FEV(1), a measure of airflow limitation, the lack of systemic markers to reflect changes in lung inflammation critically limits the testing of proposed therapeutics.
We sought to prospectively identify and validate peripheral blood leukocyte genes that could mark resolution of pulmonary infection and inflammation using a model by which RNA transcripts could increase the predictive value of spirometry.
Peripheral blood mononuclear cells were isolated from 10 patients with CF and acute pulmonary exacerbations before and after therapy. RNA expression profiling revealed that 10 genes significantly changed with treatment when compared with matched non-CF and control subjects with stable CF to establish baseline transcript abundance. Peripheral blood mononuclear cell RNA transcripts were prospectively validated, using real-time polymerase chain reaction amplification, in an independent cohort of acutely ill patients with CF (n = 14). Patients who responded to therapy were analyzed using general estimating equations and multiple logistic regression, such that changes in FEV(1)% predicted were regressed with transcript changes.
Three genes, CD64, ADAM9, and CD36, were significant and independent predictors of a therapeutic response beyond that of FEV(1) alone (P < 0.05). In both cohorts, receiver operating characteristic analysis revealed greater accuracy when genes were combined with FEV(1).
Circulating mononuclear cell transcripts characterize a response to the treatment of pulmonary exacerbations. Even in small patient cohorts, changes in gene expression in conjunction with FEV(1) may enhance current outcomes measures for treatment response.
Acute hypoxic vasoconstriction and development of hypoxic pulmonary hypertension (PHTN) are unique properties of the pulmonary circulation. The pulmonary endothelium produces vasoactive factors, ...including nitric oxide (NO), that modify these phenomena. We tested the hypothesis that NO produced by endothelial nitric oxide synthase (eNOS) modulates pulmonary vascular responses to hypoxia using mice with targeted disruption of the eNOS gene (eNOS-/-). Marked PHTN was found in eNOS-/- mice raised in mild hypoxia when compared with either controls or eNOS-/- mice raised in conditions simulating sea level. We found an approximate twofold increase in partially and fully muscularized distal pulmonary arteries in eNOS-/- mice compared with controls. Consistent with vasoconstriction being the primary mechanism of PHTN, however, acute inhalation of 25 ppm NO resulted in normalization of RV pressure in eNOS-/- mice. In addition to studies of eNOS-/- mice, the dose-effect of eNOS was tested using heterozygous eNOS+/- mice. Although the lungs of eNOS+/- mice had 50% of normal eNOS protein, the response to hypoxia was indistinguishable from that of eNOS-/- mice. We conclude that eNOS-derived NO is an important modulator of the pulmonary vascular response to chronic hypoxia and that more than 50% of eNOS expression is required to maintain normal pulmonary vascular tone.
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