Investigating the Unbinding of Muscarinic Antagonists from the Muscarinic 3 Receptor.

Patient symptom relief is often heavily influenced by the residence time of the inhibitor-target complex. For the human muscarinic receptor 3 (hMR3), tiotropium is a long-acting bronchodilator used in conditions such as asthma or chronic obstructive pulmonary disease (COPD). The mechanistic insights into this inhibitor remain unclear; specifically, the elucidation of the main factors determining the unbinding rates could help develop the next generation of antimuscarinic agents. Using our novel unbinding algorithm, we were able to investigate ligand dissociation from hMR3. The unbinding paths of tiotropium and two of its analogues, N-methylscopolamin and homatropine methylbromide, show a consistent qualitative mechanism and allow us to identify the structural bottleneck of the process. Furthermore, our machine learning-based analysis identified key roles of the ECL2/TM5 junction involved in the transition state. Additionally, our results point to relevant changes at the intracellular end of the TM6 helix leading to the ICL3 kinase domain, highlighting the closest residue L482. This residue is located right between two main protein binding sites involved in signal transduction for hMR3's activation and regulation. We also highlight key pharmacophores of tiotropium that play determining roles in the unbinding kinetics and could aid toward drug design and lead optimization.

An in silico and in vitro integrated analysis method to reveal the curative mechanisms and pharmacodynamic substances of Bufei granule on chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a common respiratory disease with high disability and mortality. Clinical studies have shown that the Traditional Chinese Medicine Bufei Granule (BFG) has conspicuous effects on relieving cough and improving lung function in patients with COPD and has a reliable effect on the treatment of COPD, whereas the therapeutic mechanism is vague. In the present study, the latent bronchodilators and mechanism of BFG in the treatment of COPD were discussed through the method of network pharmacology. Then, the molecular docking and molecular dynamics simulation were performed to calculate the binding efficacy of corresponding compounds in BFG to muscarinic receptor. Finally, the effects of BFG on bronchial smooth muscle were validated by in vitro experiments. The network pharmacology results manifested the anti-COPD effect of BFG was mainly realized via restraining airway smooth muscle contraction, activating cAMP pathways and relieving oxidative stress. The results of molecular docking and molecular dynamics simulation showed alpinetin could bind to cholinergic receptor muscarinic 3. The in vitro experiment verified both BFG and alpinetin could inhibit the levels of CHRM3 and acetylcholine and could be potential bronchodilators for treating COPD. This study provides an integrating network pharmacology method for understanding the therapeutic mechanisms of traditional Chinese medicine, as well as a new strategy for developing natural medicines for treating COPD.

Relationships of serum CC16 levels with smoking status and lung function in COPD.

BACKGROUND: The club cell secretory protein (CC16) has anti-inflammatory and antioxidant effects, and low CC16 serum levels have been associated with both risk and progression of COPD, yet the interaction between smoking and CC16 on lung function outcomes remains unknown. METHODS: Utilizing cross-sectional data on United States veterans, CC16 serum concentrations were measured by ELISA and log transformed for analyses. Spirometry was conducted and COPD status was defined by post-bronchodilator FEV1/FVC ratio < 0.7. Smoking measures were self-reported on questionnaire. Multivariable logistic and linear regression were employed to examine associations between CC16 levels and COPD, and lung function with adjustment for covariates. Unadjusted Pearson correlations described relationships between CC16 level and lung function measures, pack-years smoked, and years since smoking cessation. RESULTS: The study population (N = 351) was mostly male, white, with an average age over 60 years. An interaction between CC16 and smoking status on FEV1/FVC ratio was demonstrated among subjects with COPD (N = 245, p = 0.01). There was a positive correlation among former smokers and negative correlation among current or never smokers with COPD. Among former smokers with COPD, CC16 levels were also positively correlated with years since smoking cessation, and inversely related with pack-years smoked. Increasing CC16 levels were associated with lower odds of COPD (ORadj = 0.36, 95% CI 0.22-0.57, Padj < 0.0001). CONCLUSIONS: Smoking status is an important effect modifier of CC16 relationships with lung function. Increasing serum CC16 corresponded to increases in FEV1/FVC ratio in former smokers with COPD versus opposite relationships in current or never smokers. Additional longitudinal studies may be warranted to assess relationship of CC16 with smoking cessation on lung function among subjects with COPD.

Using Filters to Estimate Regional Lung Deposition with Dry Powder Inhalers.

PURPOSE: To develop an in vitro method to rapidly evaluate regional lung doses delivered by pharmaceutical inhalers. Currently, cascade impactor measurements are used, but these are resource intensive and require significant post processing of in vitro data to arrive at regional deposition estimates. METHODS: We present a specialized filter apparatus that mimics tracheobronchial (TB) deposition of pharmaceutical aerosols emitted by commercially available dry powder inhalers (DPIs). The filter housing includes an electrostatic neutralizer to eliminate artificial electrostatic filtration effects. Regional deposition (tracheobronchial and alveolar) for four DPIs (Onbrez Breezhaler, Flovent Diskus, Pulmicort Turbuhaler, and Asmanex Twisthaler) was estimated using cascade impactor measurements and an in silico regional deposition model. These estimates were compared to direct measurements of regional deposition as provided by the TB filter mimic and an absolute filter placed downstream of the TB filter housing, representing the alveolar dose. RESULTS: The two methods were shown to provide similar estimates of extrathoracic, tracheobronchial, and alveolar deposition, as well as total recovery of active pharmaceutical ingredients. CONCLUSIONS: Because of its design, the TB filter apparatus makes it possible to estimate regional deposition with inhalers directly using variable inhalation profiles without any additional equipment or changes to the experimental configuration. This method may be useful to expedite development of both innovative and generic drug products as it provides regional respiratory tract deposition estimates using fewer resources than exisiting methods.

Evaluation of the peripheral blood eosinophil count as a predictor for fractional exhaled nitric oxide or bronchodilator reversibility test outcome.

Objective: This study aimed to explore the usefulness of the peripheral blood eosinophil count (PBEC) in assessing the level of fractional exhaled nitric oxide (FeNO) and predicting bronchodilation test results. Methods: We retrospectively analyzed the data of 384 outpatients who underwent FeNO measurement at our Department of Respiratory and Critical Care Medicine from March to June 2019. The FeNO level was compared among different PBECs to explore the association among them. Furthermore, the sensitivity and specificity of PBECs in predicting bronchodilation test results were assessed by using receiver operating characteristic (ROC) curve analysis. Results: There was a moderate correlation between PBECs and FeNO levels (r = 0.414; p < 0.05). In the subjects with PBECs ≥ 0.3 × 109/L, the median FeNO level was 39 ppb (interquartile range, 22.5-65.5 ppb), significantly higher than in the subjects with PBECs < 0.3 × 109/L. The area under the ROC curve was 0.707 (p < 0.05). The maximum Youden index (0.348) was at PBECs = 0.205 × 109/L, which achieved sensitivity and specificity of 63% and 71.8%, respectively. Conclusion: PBECs ≥ 0.3 × 109/L can predict a positive bronchodilation test result and a high FeNO level, with a probability of 50% in the subjects with chronic cough and shortness of breath; in the absence of corresponding symptoms and a low PBEC, the predictive value was small. For hospitals not able to conduct FeNO measurements, for outpatients with poor economic conditions, and for patients with confirmed or suspected novel coronavirus disease 2019, the PBEC, in conjunction with a patient's clinical symptoms, can improve the diagnostic accuracy of allergic asthma and assessment of airway inflammation while reducing the risk of infection.

Investigation of drug dissolution and uptake from low-density DPI formulations in an impactor-integrated cell culture model.

Besides deposition, pulmonary bioavailability is determined by dissolution of particles in the scarce epithelial fluid and by cellular API uptake. In the present work, we have developed an experimental in vitro model, which is combining the state-of-the-art next generation impactor (NGI), used for aerodynamic performance assessment of inhalation products, with a culture of human alveolar A549 epithelial cells to study the fate of inhaled drugs following lung deposition. The goal was to investigate five previously developed nano-milled and spray-dried budesonide formulations and to examine the suitability of the in vitro test model. The NGI dissolution cups of stages 3, 4, and 5 were transformed to accommodate cell culture inserts while assuring minimal interference with the air flow. A549 cells were cultivated at the air-liquid interface on Corning® Matrigel® -coated inserts. After deposition of aerodynamically classified powders on the cell cultures, budesonide amount was determined on the cell surface, in the interior of the cell monolayer, and in the basal solution for four to eight hours. Significant differences in the total deposited drug amount and the amount remaining on the cell surface at the end of the experiment were found between different formulations and NGI stages. Roughly 50% of budesonide was taken up by the cells and converted to a large extent to its metabolic conjugate with oleic acid for all formulations and stages. Prolonged time required for complete drug dissolution and cell uptake in case of large deposited powder amounts suggested initial drug saturation of the surfactant layer of the cell surface. Discrimination between formulations with respect to time scale of dissolution and cell uptake was possible with the present test model providing useful insights into the biopharmaceutical performance of developed formulations that may be relevant for predicting local bioavailability. The absolute quantitative result of cell uptake and permeation into the systemic compartment is unreliable, though, because of partly compromised cell membrane integrity due to particle impaction and professed leakiness of A549 monolayer tight junctions, respectively.

[Extracellular molecules controlling the contraction of airway smooth muscle and their potential contribution to bronchial hyperresponsiveness]. / La contractilité du muscle lisse des voies respiratoires est régulée par de nombreuses molécules extracellulaires qui pourraient contribuer à l'hyperréactivité bronchique.

INTRODUCTION: A significant portion of symptoms in some lung diseases results from an excessive constriction of airways due to the contraction of smooth muscle and bronchial hyperresponsiveness. A better understanding of the extracellular molecules that control smooth muscle contractility is necessary to identify the underlying causes of the problem. STATE OF KNOWLEDGE: Almost a hundred molecules, some of which newly identified, influence the contractility of airway smooth muscle. While some molecules activate the contraction, others activate the relaxation, thus acting directly as bronchoconstrictors and bronchodilators, respectively. Other molecules do not affect contraction directly but rather influence it indirectly by modifying the effect of bronchoconstrictors and bronchodilators. These are called bronchomodulators. Some of these bronchomodulators increase the contractile effect of bronchoconstrictors and could thus contribute to bronchial hyperresponsiveness. PROSPECTS: Considering the high number of molecules potentially involved, as well as the level of functional overlap between some of them, identifying the extracellular molecules responsible for excessive airway constriction in a patient is a major contemporary challenge.

Suitability and functional characterization of two Calu-3 cell models for prediction of drug permeability across the airway epithelial barrier.

The Calu-3 cell line has been largely investigated as a physiological and pharmacological model of the airway epithelial barrier. Its suitability for prediction of drug permeability across the airway epithelia, however, has not been yet evaluated by using large enough set of model drugs. We evaluated two Calu-3 cell models (air-liquid and liquid-liquid) for drug permeability prediction based on the recent regulatory guidelines on showing suitability of in vitro permeability methods for drug permeability classification. Bidirectional permeability assays using 22 model drugs and several zero permeability markers, as well as using ABC transporter substrates were conducted. Functional activity of P-gp, but not of BCRP was revealed. The potential of the Calu-3 cells to be used as a model of the nasal epithelial barrier, despite their different anatomical origin, has been demonstrated by the obtained excellent correlation with the fully differentiated 3D human nasal epithelial model (MucilAir™) for 11 model drugs, as well as by the good correlation obtained with the human nasal epithelial cell line RPMI 2650. In addition, the permeability values determined in the two Calu-3 models correlated well with the intestinal permeability model Caco-2.

Folium Sennae and emodin reverse airway smooth muscle contraction.

The objective of this project was to find a bronchodilatory compound from herbs and clarify the mechanism. We found that the ethanol extract of Folium Sennae (EEFS) can relax airway smooth muscle (ASM). EEFS inhibited ASM contraction, induced by acetylcholine, in mouse tracheal rings and lung slices. High-performance liquid chromatography assay showed that EEFS contained emodin. Emodin had a similar reversal action. Acetylcholine-evoked contraction was also partially reduced by nifedipine (a selective inhibitor of L-type voltage-dependent Ca2+ channels, LVDCCs), YM-58483 (a selective inhibitor of store-operated Ca2+ entry, SOCE), as well as Y-27632 (an inhibitor of Rho-associated protein kinase). In addition, LVDCC- and SOCE-mediated currents and cytosolic Ca2+ elevations were inhibited by emodin. Emodin reversed acetylcholine-caused increases in phosphorylation of myosin phosphatase target subunit 1. Furthermore, emodin, in vivo, inhibited acetylcholine-induced respiratory system resistance in mice. These results indicate that EEFS-induced relaxation results from emodin inhibiting LVDCC, SOCE, and Ca2+ sensitization. These findings suggest that Folium Sennae and emodin may be new sources of bronchodilators.

Airway smooth muscle adapting in dynamic conditions is refractory to the bronchodilator effect of a deep inspiration.

Airway smooth muscle (ASM) is continuously strained during breathing at tidal volume. Whether this tidal strain influences the magnitude of the bronchodilator response to a deep inspiration (DI) is not clearly defined. The present in vitro study examines the effect of tidal strain on the bronchodilator effect of DIs. ASM strips from sheep tracheas were mounted in organ baths and then subjected to stretches (30% strain), simulating DIs at varying time intervals. In between simulated DIs, the strips were either held at a fixed length (isometric) or oscillated continuously by 6% (length oscillations) to simulate tidal strain. The contractile state of the strips was also controlled by adding either methacholine or isoproterenol to activate or relax ASM, respectively. Although the time-dependent gain in force caused by methacholine was attenuated by length oscillations, part of the acquired force in the oscillating condition was preserved postsimulated DIs, which was not the case in the isometric condition. Consequently, the bronchodilator effect of simulated DIs (i.e., the decline in force postsimulated versus presimulated DIs) was attenuated in oscillating versus isometric conditions. These findings suggest that an ASM operating in a dynamic environment acquired adaptations that make it refractory to the decline in contractility inflicted by a larger strain simulating a DI.

Bronchodilation induced by PGE2 is impaired in Group III pulmonary hypertension.

BACKGROUND AND PURPOSE: In patients with pulmonary hypertension (PH) associated with lung disease and/or hypoxia (Group III), decreased pulmonary vascular tone and tissue hypoxia is therapeutically beneficial. PGE2 and PGI2 induce potent relaxation of human bronchi from non-PH (control) patients via EP4 and IP receptors, respectively. However, the effects of PGE2 /PGI2 and their mimetics on human bronchi from PH patients are unknown. Here, we have compared relaxant effects of several PGI2 -mimetics approved for treating PH Group I with several PGE2 -mimetics, in bronchial preparations derived from PH Group III and control patients. EXPERIMENTAL APPROACH: Relaxation of bronchial muscle was assessed in samples isolated from control and PH Group III patients. Expression of prostanoid receptors was analysed by western blot and real-time PCR, and endogenous PGE2 , PGI2 , and cAMP levels were determined by ELISA. KEY RESULTS: Maximal relaxations induced by different EP4 receptor agonists (PGE2 , L-902688, and ONO-AE1-329) were decreased in human bronchi from PH patients, compared with controls. However, maximal relaxations produced by PGI2 -mimetics (iloprost, treprostinil, and beraprost) were similar for both groups of patients. Both EP4 and IP receptor protein and mRNA expressions were significantly lower in human bronchi from PH patients. cAMP levels significantly correlated with PGI2 but not with PGE2 levels. CONCLUSION AND IMPLICATIONS: The PGI2 -mimetics retained maximal bronchodilation in PH Group III patients, whereas bronchodilation induced by EP4 receptor agonists was decreased. Restoration of EP4 receptor expression in airways of PH Group III patients with respiratory diseases could bring additional therapeutic benefit.

The Respimat® Soft Mist Inhaler: Implications of Drug Delivery Characteristics for Patients.

Successful treatment for respiratory diseases relies on effective delivery of medication to the lungs using an inhalation device. Different inhalers have distinct characteristics affecting drug administration and patient adherence, which can impact clinical outcomes. We report on the development of the Respimat® soft mist inhaler (SMI) and compare key attributes with metered-dose inhalers (MDIs) and dry powder inhalers (DPIs). The Respimat SMI, a pocket-sized device generating a single-breath, inhalable aerosol, was designed to enhance drug delivery to the lungs, reduce the requirements for patient coordination and inspiratory effort, and improve the patients' experience and ease of use. The drug deposition profile with Respimat SMI is favorable compared with MDIs and DPIs, with higher drug deposition to the lung and peripheral airways. The slow velocity and long spray duration of the Respimat SMI aerosol also aid patient coordination. Clinical equivalence has been demonstrated for maintenance treatment of chronic obstructive pulmonary disease using once-daily tiotropium between Respimat SMI (5 µg) and HandiHaler DPI (18 µg). In comparative studies, patients preferred Respimat SMI to MDIs and DPIs; they reported that Respimat SMI was easy to use and felt the inhaled dose was delivered. The Respimat SMI, designed to generate a slow-moving and fine mist, is easy to use and effectively delivers drug treatment to the lungs. The patient-centered design of Respimat SMI improved patient satisfaction, and may help to promote long-term adherence and improve clinical outcomes with asthma and chronic obstructive pulmonary disease.

Evaluation of systemic absorption and bronchodilator effect of glycopyrronium bromide delivered by nebulizer or a dry powder inhaler in subjects with chronic obstructive pulmonary disease.

BACKGROUND: Effective bronchodilator therapy depends upon adequate drug deposition in the lung. COPD patients who are unable to administer medications efficiently with conventional inhalers may benefit from the use of a nebulizer device. The aim of this study was to evaluate the systemic bioavailability and bronchodilator response of glycopyrronium bromide (GLY) administered by a novel nebulizer (eFlow® closed system [CS] vibrating membrane nebulizer) or dry powder inhaler (DPI) in subjects with moderate-to-severe chronic obstructive pulmonary disease (COPD). METHODS: In this randomized, open-label, single-dose, five-way crossover study, subjects received a sequence of either 50 µg GLY delivered by eFlow CS nebulizer (GLY/eFlow) or 63 µg GLY delivered by DPI (GLY/DPI), with and without activated charcoal, followed by intravenous infusion of 50 µg GLY with a washout period of 7 days between doses. Endpoints included plasma pharmacokinetics, safety and efficacy. RESULTS: The mean (± SD) baseline predicted forced expiratory volume in 1 s (FEV1) of the 30 subjects who completed the study was 51 ± 15%, with a FEV1/forced vital capacity ratio of 50 ± 11%. Without charcoal, the absolute systemic bioavailability of GLY/eFlow and GLY/DPI were approximately 15 and 22%, respectively. Changes from baseline in FEV1 at 60 min post-dose, without administration of charcoal, were 0.180 L and 0.220 L for GLY/eFlow and GLY/DPI, respectively; FEV1 improvements were similar when charcoal was administered (0.220 L for both GLY/eFlow and GLY/DPI). There were no significant differences in spirometry between the two devices. Fewer subjects administered GLY/eFlow reported adverse events (n = 15) than GLY/DPI (n = 18). CONCLUSIONS: After single doses, GLY/DPI delivered numerically higher peak and steady state levels of drug than did GLY/eFlow. Nebulized GLY produced similar bronchodilation but lower systemic levels of drug than GLY/DPI. Slightly higher number of subjects reported adverse events with GLY/DPI than with GLY/eFlow. Nebulized GLY may offer an effective alternative to patients with COPD not adequately treated with other devices. TRIAL REGISTRATION: NCT02512302 (ClinicalTrials.gov). Registered 28 May 2015.

In vitro investigation on the impact of airway mucus on drug dissolution and absorption at the air-epithelium interface in the lungs.

Although the mucus layer is the first biological barrier encountered by inhaled drugs upon their deposition in the upper airways, its potential impact on drug dissolution and absorption in the lung has hardly been investigated. Bio-relevant in vitro models were therefore used to assess the role of airway mucus in the fate of drug particles at the air-epithelium interface. Salbutamol and indomethacin were used as model Biopharmaceutics Classification System (BCS) class III and class II drugs, respectively. Dry powders were reproducibly aerosolised using a PennCentury™ Dry Powder Insufflator onto multiple air-liquid interfaced layers of the broncho-epithelial cell line Calu-3 or thin layers of porcine tracheal mucus mounted onto Transwells® inserts, as well as on empty Transwells®. Comparison of the permeation profiles of the two drugs indicated that mucus acted as a barrier for salbutamol transport but increased that of indomethacin, suggesting it facilitates the dissolution of poorly soluble drugs. In presence of Calu-3 layers, the permeability of salbutamol was even more restricted while indomethacin transport was enhanced further. This study demonstrates mucus distinctly affects the absorption characteristics of drugs with different physico-chemical properties. Hence, drug-mucus interactions should be considered during the development of inhaled drugs.

Recent advances in aerosolised drug delivery.

Pulmonary route is extensively studied for the diagnosis and treatment of pulmonary and extra pulmonary disease conditions such as asthma, tuberculosis, emphysema, and bronchitis. Formulation design, inhalation device and particle size play key role in determining the aerosol performance. The lack of desired clinical outcome along with the problem regarding efficacy or any adverse drug effect may arise due to improper training and education in use of the device to control the actuation and aerosol inhalation. This review summarizes the difference in the mechanistic features of current marketed aerosol delivery devices with respect to mechanism of aerosol generation with possible advancements in the aerosol design. The delivery options in the pulmonary route and its merits together with the limitations are also discussed. An update is provided regarding the current research and clinical outcome of the use of inhalational technology.

Design, synthesis and biological evaluation of 5-(2-amino-1-hydroxyethyl)-8-hydroxyquinolin-2(1H)-one derivatives as potent ß2-adrenoceptor agonists.

A series of novel ß2-adrenoceptor agonists with a 5-(2-amino-1-hydroxyethyl)-8-hydroxyquinolin-2(1H)-one moiety was designed, synthesized and evaluated for biological activity in human embryonic kidney 293 cells and isolated guinea pig trachea. Compounds 9g and (R)-18c exhibited the most excellent ß2-adrenoceptor agonistic effects and high ß2/ß1-selectivity with EC50 values of 36 pM for 9g and 21 pM for (R)-18c. They produced potent airway smooth muscle relaxant effects with fast onset of action and long duration of action in an in vitro guinea pig trachea model of bronchodilation. These results support further development of the two compounds into drug candidates.

The Role of FENO in Comparison to Spirometry and ACT in Control of Children Asthma Symptoms.

Fractional exhaled nitric oxide (FeNO) is a noninvasive marker of inflammation, used for monitoring asthma. The aim of this study was to compare FeNO, asthma control test (ACT), and lung function test (spirometry) in children aged 8-15 years. This observational, cross-sectional study was performed on76 asthmatic children (age, 8-15 years), who were referred to the Department of Immunology and Allergy, Children's Medical Center, Tehran, Iran during 2012-2013. Patients were matched for sex and age. The recruited patients were selected via consecutive sampling. FeNO was measured with a portable electrochemical analyzer and forced spirometry was performed according to the American Thoracic Society (ATS) guidelines. The ACT questionnaire was used and completed for all the patients. The mean FeNO was 28.5±29.1 ppb, and the mean ACT score was 19.8±3.6. FeNO was significantly correlated with forced expiratory volume (FEV1) (r, 0.232; p=0.049) or 25-75% maximum expiratory flow (MEF 25-75) (r, -0.304; p=0.009). FeNO showed no significant correlation with ACT score or FEV1/forced vital capacity (FVC) (p>0.05). Additionally, there was no significant correlation between FeNO and changes in FEV1 and MEF 25-75% before and after the administration of bronchodilators (p>0.05). To improve asthma control, childhood ACT, FeNO, and spirometric tests can be used as complementary tools in clinical practice to detect children with poorly controlled asthma.

Effects of Formulation Variables on Lung Dosimetry of Albuterol Sulfate Suspension and Beclomethasone Dipropionate Solution Metered Dose Inhalers.

The performance of pressurized metered dose inhalers (MDIs) is affected by formulation and device variables that impact delivered dose, aerodynamic particle size distribution, and consequently lung deposition and therapeutic effect. Specific formulation variables of relevance to two commercially available products-Proventil® HFA [albuterol sulfate (AS) suspension] and Qvar® [beclomethasone dipropionate (BDP) solution]-were evaluated to determine their influence on key performance attributes measured experimentally with in vitro cascade impaction studies. These commercial MDIs, utilized as model systems, provided mid-points for a design of experiments (DoE) plan to manufacture multiple suspension and solution MDI formulations. The experimental results were utilized as input variables in a computational dosimetry model to predict the effects of MDI formulation variables on lung deposition. For the BDP solution DoE MDIs, increased concentrations of surfactant oleic acid (0-2% w/w) increased lung deposition from 24 to 46%, whereas changes in concentration of the cosolvent ethanol (7-9% w/w) had no effect on lung deposition. For the AS suspension DoE MDIs, changes in oleic acid concentration (0.005-0.25% w/w) did not have significant effects on lung deposition, whereas lung deposition decreased from 48 to 26% as ethanol concentration increased from 2 to 20% w/w, and changes in micronized drug volumetric median particle size distribution (X50, 1.4-2.5 µm) increased deposition in the tracheobronchial airways from 5 to 11%. A direct correlation was observed between fine particle fraction and predicted lung deposition. These results demonstrate the value of using dosimetry models to further explore relationships between performance variables and lung deposition.

Interaction of DNA and mononucleotides with theophylline investigated using electrochemical biosensors and biosensing.

The understanding of DNA-drug interaction mechanism is among the important aspects of biological studies for drug design, discovery and pharmaceutical development processes. Published rather detailed FTIR and UV-visible spectroscopic studies on the interactions of theophylline, theobromine and caffeine with calf thymus DNA have shown effective binding of these methylxanthine derivatives to DNA and RNA involving H-bonds. However, to our knowledge, there is no such investigation using electrochemical approach. As a novelty of the study, in this paper the bioelectrochemical approach has been chosen for the investigation of an interaction of low molecular salmon sperm dsDNA, ssDNA and mononucleotides with theophylline (TP) in aqueous phosphate buffered medium using DNA-based electrochemical biosensors and biosensing in solution phase. Exploitation of the electrochemical approach via changes in square wave voltammetric responses of deoxyguanosine (dGuo) and deoxyadenosine (dAdo) provided a new indication on preferential association of TP with dGuo in the case of double helical dsDNA structure which was not reported previously. Moreover, an attachment of TP molecules outside DNA was found in the presence of high concentration of 3.3 × 10-4 M TP in solution which diminishes the electron transfer and leads to the difficulties in quantitative evaluation of the TP and dGuo voltammetric responses. The changes in UV-vis and FTIR spectra obtained in the same medium confirmed the association interaction of TP with both nucleobases. Utilizing the model and the published energies of hydrogen bonding stabilization, the formation of a DNA-TP complex was predicted through the intermolecular H-bonds between TP and the NH-CO moiety of guanine and the N-NH2 moiety of adenine.

Uncovering the regional localization of inhaled salmeterol retention in the lung.

Treatment of respiratory disease with a drug delivered via inhalation is generally held as being beneficial as it provides direct access to the lung target site with a minimum systemic exposure. There is however only limited information of the regional localization of drug retention following inhalation. The aim of this study was to investigate the regional and histological localization of salmeterol retention in the lungs after inhalation and to compare it to systemic administration. Lung distribution of salmeterol delivered to rats via nebulization or intravenous (IV) injection was analyzed with high-resolution mass spectrometry imaging (MSI). Salmeterol was widely distributed in the entire section at 5 min after inhalation, by 15 min it was preferentially retained in bronchial tissue. Via a novel dual-isotope study, where salmeterol was delivered via inhalation and d3-salmeterol via IV to the same rat, could the effective gain in drug concentration associated with inhaled delivery relative to IV, expressed as a site-specific lung targeting factor, was 5-, 31-, and 45-fold for the alveolar region, bronchial sub-epithelium and epithelium, respectively. We anticipate that this MSI-based framework for quantifying regional and histological lung targeting by inhalation will accelerate discovery and development of local and more precise treatments of respiratory disease.