Elsevier

Biochemical Pharmacology

Volume 82, Issue 3, 1 August 2011, Pages 248-259
Biochemical Pharmacology

Formation of new bioactive organic nitrites and their identification with gas chromatography–mass spectrometry and liquid chromatography coupled to nitrite reduction

https://doi.org/10.1016/j.bcp.2011.04.005Get rights and content

Abstract

Nitric oxide (NO) donors, notably organic nitrates and nitrites are used therapeutically but tolerance develops rapidly, making the use of e.g. nitroglycerin difficult. NO donation in the pulmonary vascular bed might be useful in critically ill patients. Organic nitrites are not associated with tachyphylaxis but may induce methaemoglobinemia and systemic hypotension which might hamper their use. We hypothesised that new lung-selective NO donors can be identified by utilizing exhaled NO as measure for pulmonary NO donation and systemic arterial pressure to monitor hypotension and tolerance development. Solutions of alcohols and carbohydrates were reacted with NO gas and administered to ventilated rabbits for evaluation of in vivo NO donation. Chemical characterization was made by liquid chromatography with on-line nitrite reduction (LC-NO) and by gas chromatography–mass spectrometry (GC–MS). In vivo experiments showed that the hydroxyl-containing compounds treated with NO gas yielded potent NO donors, via nitrosylation to organic nitrites. Analyses by LC-NO showed that the reaction products were able to release NO in vitro. In GC–MS the reaction products were determined to be the organic nitrites, where some are new chemical entities. Non-polar donors preferentially increased exhaled NO with less effect on systemic blood pressure whereas more polar molecules had larger effects on systemic blood pressure and less on exhaled NO. We conclude that new organic nitrites suitable for intravenous administration are produced by reacting NO gas and certain hydroxyl-containing compounds in aqueous solutions. Selectivity of different organic nitrites towards the pulmonary and systemic circulation, respectively, may be determined by molecular polarity.

Introduction

Nitric oxide donors are used to supply a patient with exogenous nitric oxide (NO) and can be used in conditions and diseases where increased NO is thought to be beneficial. There is a wide range of NO donors with different chemistry and biochemistry available for experimental use [1]. The list of conditions and diseases is extensive for experimental studies that have suggested beneficial effects, but only a few compounds are used in clinical practice [2]. Nitroglycerin and isosorbide mononitrate are used in acute and chronic anginal pain respectively and nitroglycerin may be used in the treatment of anal fissure [2]. Isosorbide dinitrate together with hydralazine is used for heart failure in African-Americans [2]. Inhaled amyl nitrite has been used in anginal pain and as an antidote in life-threatening cyanide intoxication [3]. Organic nitrates and organic nitrites are converted to NO in vivo which can be seen as increased exhaled NO and decreased systemic blood pressure [4], [5]. A significant clinical problem with organic nitrates is the rapid tolerance development which can be demonstrated experimentally with decreased generation of NO to exhaled air [6], [7]. Organic nitrites exhibit little or no tolerance [8], and recently ethyl nitrite has been suggested as an inhaled therapeutic [9]. However, the use of ethyl nitrite and other organic nitrites has been questioned due to significant methaemoglobin formation and other serious adverse effects [10], [11]. Sodium nitroprusside is used in hypertensive crises but is associated with tolerance development and risk of cyanide intoxication and its use is sometimes therefore discouraged [12]. Inhaled NO is used in persistent pulmonary hypertension of the newborn [13] whereas its use in acute lung injury and acute respiratory distress syndrome is not recommended [14]. Other conditions where treatment with exogenous NO has been suggested from experimental studies include myocardial ischemia reperfusion [15], [16], splanchnic ischemia reperfusion and acute intestinal inflammation [17], stroke [18], pulmonary hypertension [19] and sepsis [20]. The reasons for the lack of development of new NO donors that actually reach clinical practice are complex and may include side-effects in the form of systemic hypotension, headache, lack of specificity, tolerance development and toxicity [2]. Altogether there is a quest for new NO donors [2]. We believe that there is a specific need for intravenous NO donors with selectivity for the pulmonary circulation and that can be used, for example, in acute pulmonary embolism, where recent data indicate a protective role of endogenous NO [21]. We hypothesised that new lung-selective NO donors can be identified by utilizing exhaled NO as measure for pulmonary NO donation and systemic arterial pressure to monitor hypotension and tolerance development. We find that NO donors are formed, in a solution suitable for intravenous delivery, when deoxygenated aqueous solutions of alcohols and carbohydrates are reacted with NO gas and that these NO donors have different selectivity towards the pulmonary and systemic circulations.

Section snippets

Preparation of reaction products between compounds and nitric oxide gas

Aqueous solutions of alcohols, sugars and related compounds (please refer to Table 1 and supplementary Table A.1) were prepared and placed in gas tight cylinders (volume 20–150 ml) with taps in both ends and a sideway port with a septum plug (F-145, Alltech Biotechnology, Ireland) in the middle portion. The cylinders were filled to 75% with solution, allowing a gaseous head-space. The solutions were deoxygenated by means of helium bubbling for 10 min and purged with pure nitric oxide gas for 3 

NO treated lipid emulsion and glycerol infused intravenously in vivo

Initial experiments showed that bolus infusions intravenously (0.5–2.5 ml) of a lipid emulsion (Intralipid) saturated with pure NO gas transiently increased FENO paralleled with a transient drop in MAP (data not shown). These responses could not be reproduced by only the native lipid emulsion (data not shown). In another experiment a rabbit with inhibited endogenous NO production (l-NAME 30 mg kg−1) first for 30 min intravenously received deoxygenated saline equilibrated with NO gas (0.5 ml kg−1 min−1

Discussion

For more than a century, organic nitrites have been used as volatile NO donors for relieving anginal pain. Further use has been limited due to problems with administration, dosing and adverse effects.

The present study introduces a method for producing potent NO donor solutions, evidenced by increases in exhaled NO and decreases of systemic blood pressure and by the ability to deliver NO gas in a nitrite reduction system after HPLC. The NO donor molecules were identified to be organic nitrites

Conflicts of interest statement

The authors (except ML) have filed patent applications on organic nitrites. LEG is a minority shareholder and member of the board of directors of Aerocrine AB (publ.) and is an inventor of patents concerning the use of exhaled nitric oxide.

Acknowledgements and role of funding sources

This study was supported by the European Space Agency, the Swedish National Space Board, Fraenckel's Foundation for Medical Research, the Lars Hierta Foundation, the Swedish Science Council project 07919, the Swedish Heart-Lung Foundation and the Karolinska Institutet (MD/OD/PhD program grant). The funding sources solely provided financial support and were not involved in any part of the conduct of the research.

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