Impact of breath sample collection method and length of storage of breath samples in Tedlar bags on the level of selected volatiles assessed using gas chromatography-ion mobility spectrometry (GC-IMS).

The analysis of volatile organic compounds (VOCs) in exhaled air has attracted the interest of the scientific community because it provides the possibility of monitoring physiological and metabolic processes and non-invasive diagnostics of various diseases. However, this method remains underused in clinical practice as well as in research because of the lack of standardized procedures for the collection, storage and transport of breath samples, which would guarantee good reproducibility and comparability of results. The method of sampling, as well as the storage time of the breath samples in the polymer bags used for sample storage and transport, affect the composition and concentration of VOCs present in the breath samples. The aim of our study was to compare breath samples obtained using two methods with fully disposable equipment: a Haldane sampling tube intended for direct breath collection and breath samples exhaled into a transparent Tedlar bag. The second task was to monitor the stability of selected compounds of real breath samples stored in a Tedlar bag for 6 h. Gas chromatography coupled with ion mobility spectrometry (GC-IMS) implemented in the BreathSpec®device was used to analyse exhaled breath. Our results showed a significant difference in the signal intensity of some volatiles when taking a breath sample with a Haldane tube and a Tedlar bag. Due to its endogenous origin, acetone levels were significantly higher when the Haldane tube sampler was used while elevated levels of 2-propanol and unidentified VOC (designated as VOC 3) in the Tedlar bag samples likely originated from contamination of the Tedlar bags. The VOC stability study revealed compound-specific signal intensity changes of the selected VOCs with storage time in the Tedlar bags, with some volatiles showing increasing signal intensity during storage in Tedlar bags. This limits the use of Tedlar bags only for very limited time and carefully selected purpose. Our results highlight the importance of careful design and implementation of experiments and clinical protocols to obtain relevant and reliable results.

Determination of Phosphodiesterase Inhibitors Tadalafil, Roflumilast and Roflumilast N-Oxide Using LC-MS in Guinea Pig Plasma.

Phosphodiesterases are known as a super-family of 11 isoenzymes, which can exert various functions based on their organ distribution. In this work, a rapid and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry method was developed for quantification of tadalafil (phosphodiesterase five inhibitor), roflumilast (RF) (phosphodiesterase four inhibitor) and its active metabolite, RF N-oxide in guinea pig plasma. Chromatographic separation was carried out on UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm) at a flow rate 0.5 mL/min, using 0.2% formic acid in acetonitrile and 0.2% formic acid in water as mobile phases within 4 min. Detection was performed using a triple quadrupole mass spectrometer employing electrospray ionization operated in positive mode using multiple reaction monitoring mode. The method utilized deuterium labeled internal standards, and was validated according to European Medicines Agency guidelines. It showed excellent linearity in the range of 0.5-500.0 ng/mL for all analytes with coefficient of determination >0.99. The intra- and inter-day precisions (relative standard deviation %) were within 6.7%, and the recoveries were greater than 73.4%. Using this method, plasma samples from experiments of phosphodiesterase four, and five inhibitors in a model of ovalbumin-induced allergic inflammation in guinea pigs were analyzed.

Inhibitors of phosphodiesterases in the treatment of cough.

A group of 11 enzyme families of metalophosphohydrolases called phosphodiesterases (PDEs) is responsible for a hydrolysis of intracellular cAMP and cGMP. Xanthine derivatives (methylxanthines) inhibit PDEs without selective action on their single isoforms and lead to many pharmacological effects, e.g. bronchodilation, anti-inflammatory and immunomodulating effects, and thus they can modulate the cough reflex. Contrary, selective PDE inhibitors have been developed to inhibit PDE isoforms with different pharmacological effects based on their tissue expression. In this paper, effects of non-selective PDE inhibitors (e.g. theophylline) are discussed, with a description of other putative mechanisms in their effects on cough. Antitussive effects of selective inhibitors of several PDE isoforms are reviewed, focusing on PDE1, PDE3, PDE4, PDE5 and PDE7. The inhibition of PDEs suggests participation of bronchodilation, suppression of TRPV channels and anti-inflammatory action in cough suppression. Selective PDE3, PDE4 and PDE5 inhibitors have demonstrated the most significant cough suppressive effects, confirming their benefits in chronic inflammatory airway diseases associated with bronchoconstriction and cough.

Dose dependent effects of tadalafil and roflumilast on ovalbumin-induced airway hyperresponsiveness in guinea pigs.

INTRODUCTION: Chronic obstructive diseases of airways associated with cough and/or airway smooth muscle hyperresponsiveness are usually treated with bronchodilating and anti-inflammatory drugs. Recently, selective phosphodiesterase (PDE) 4 inhibitors have been introduced into the therapy of chronic obstructive pulmonary disease. Several studies have demonstrated their ability to influence the airway reactivity and eosinophilic inflammation by increasing the intracellular cAMP concentrations also in bronchial asthma. Furthermore, the expression of PDE5 in several immune cells suggests perspectives of PDE5 inhibitors in the therapy of inflammation, as well. PURPOSE: The aim of this study was to assess the dose-dependent effects of PDE4 and PDE5 inhibitors in allergic inflammation. Therefore, the effects of 7-days administration of PDE4 inhibitor roflumilast and PDE5 inhibitor tadalafil at two different doses in experimentally-induced allergic inflammation were evaluated. MATERIALS AND METHODS: In the study, male adult guinea pigs were used. Control group was non-sensitized. Other animals were sensitized with ovalbumin over two weeks and thereafter treated intraperitoneally for 7 days with roflumilast or tadalafil (daily dose 0.5 mg/kg or 1.0 mg/kg b.w.), or with vehicle. RESULTS: Both roflumilast and tadalafil reduced specific airway resistance after nebulization of histamine (marker of in vivo airway reactivity) at both doses used. The in vitro airway reactivity to cumulative doses of acetylcholine was significantly reduced for roflumilast at higher dose, predominantly in the lung tissue strips. Histamine-induced contractile responses were significantly influenced in both lung and tracheal tissue strips, predominantly at the higher doses. Tadalafil led to a decrease in contractile responses induced by both acetylcholine and histamine, with more significant effects in the lung tissue strips. These changes were associated with decreased numbers of circulating leukocytes and eosinophils and concentrations of interleukin (IL)-4, IL-5 and TNF-α in the lung homogenate. CONCLUSIONS: The selective PDE4 and PDE5 inhibitors alleviated allergic airway inflammation, with more significant effects at the higher doses.