Abstract
β2-Agonists are the most effective bronchodilators for the rapid relief of asthma symptoms, but for unclear reasons, their effectiveness may be decreased during severe exacerbations. Because peroxidase activity and nitrogen oxides are increased in the asthmatic airway, we examined whether salbutamol, a clinically important β2-agonist, is subject to potentially inactivating nitration. When salbutamol was exposed to myeloperoxidase, eosinophil peroxidase or lactoperoxidase in the presence of hydrogen peroxide (H2O2) and nitrite (NO2−), both absorption spectroscopy and mass spectrometry indicated formation of a new metabolite with features expected for the nitrated drug. The new metabolites showed an absorption maximum at 410 nm and pKa of 6.6 of the phenolic hydroxyl group. In addition to nitrosalbutamol (m/z 285.14), a salbutamol-derived nitrophenol, formed by elimination of the formaldehyde group, was detected (m/z 255.13) by mass spectrometry. It is noteworthy that the latter metabolite was detected in exhaled breath condensates of asthma patients receiving salbutamol but not in unexposed control subjects, indicating the potential for β2-agonist nitration to occur in the inflamed airway in vivo. Salbutamol nitration was inhibited in vitro by ascorbate, thiocyanate, and the pharmacological agents methimazole and dapsone. The efficacy of inhibition depended on the nitrating system, with the lactoperoxidase/H2O2/NO2− being the most affected. Functionally, nitrated salbutamol showed decreased affinity for β2-adrenergic receptors and impaired cAMP synthesis in airway smooth muscle cells compared with the native drug. These results suggest that under inflammatory conditions associated with asthma, phenolic β2-agonists may be subject to peroxidase-catalyzed nitration that could potentially diminish their therapeutic efficacy.
Footnotes
This study was partially supported by a Veteran Administration Merit Review Grant and a Research Initiative Project, the Cystic Fibrosis Foundation, and the National Institutes of Health National Heart, Lung, and Blood Institute [Grant HL092121].
The contents of this article do not represent the views of the Department of Veterans Affairs or the United States Government.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.170027.
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ABBREVIATIONS:
- β2-AR
- β2-adrenergic receptor
- AA
- ascorbic acid
- EBC
- exhaled breath condensate
- EPO
- eosinophil peroxidase
- LPO
- lactoperoxidase
- MPO
- myeloperoxidase
- NaSCN
- sodium thiocyanate
- EOS
- eosinophil
- PMN
- neutrophil
- DTPA
- diethylenetriamine pentaacetic acid
- MS/MS
- tandem mass spectrometry
- FT
- Fourier transform
- ICR
- ion cyclotron resonance
- CYP
- cyanopindolol
- HPLC
- high-performance liquid chromatography
- ppb
- parts per billion.
- Received May 5, 2010.
- Accepted October 19, 2010.
- U.S. Government work not protected by U.S. copyright
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