The aim of this study was to compare the binding profile of a range of β2-adrenoceptor (β2-AR) agonists and antagonists in human lung tissue. Radioligand saturation and competition binding experiments were performed by filtration with a β2-AR antagonist ([3H]propranolol) or agonist ([3H]vilanterol) radioligand and membrane fragments generated from lung parenchyma in the presence of 100 μM guanosine 5’-[β,γ-imido]triphosphate (Gpp(NH)p). In membranes prepared from human lung parenchyma, carmoterol, formoterol, ICI118551, propranolol and salbutamol resulted in inhibition of [3H]vilanterol binding to levels that were significantly different from indacaterol, salmeterol and vilanterol (ANOVA, Bonferroni post-test, P < 0.001 except formoterol vs indacaterol where P < 0.01). Indacaterol and salmeterol resulted in inhibition of [3H]vilanterol binding to levels that were not significantly different from vilanterol (ANOVA, Bonferroni post-test, P > 0.05). Indacaterol, salmeterol and vilanterol resulted in full inhibition of [3H]propranolol binding to levels not significantly different from ICI118551 (ANOVA, Bonferroni post-test, P > 0.05). Indacaterol, salmeterol and vilanterol bind to an additional site in human lung parenchyma membranes that is distinct from the β2-AR.
Inhaled β2-adrenoceptor (β2-AR) agonists are used in the treatment of both asthma [
The mechanism by which both the established (formoterol and salmeterol) and recently developed (indacaterol, olodaterol and vilanterol) LABAs achieve their long duration of action has never been fully elucidated despite almost 20 years of literature dedicated to it. A number of hypotheses have been put forward that include the “microkinetic” theory [
As part of the vilanterol drug development programme a radiolabelled form of this LABA was generated to determine its β2-AR binding characteristics in recombinant systems [
Indacaterol, salbutamol, salmeterol and vilanterol (
Non-diseased human lungs from organ donors were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA, USA) in accordance with local human biological sample management procedures. The human biological samples were sourced ethically and
their research use was in accord with the terms of the informed consents. 5 - 10 g samples of human lung parenchyma tissue obtained from 2 donors were dissected and cleaned of adherent connective and fatty tissue. Tissue samples were suspended in ice-cold assay buffer (50 mM Tris, 154 mM NaCl, 10 mM MgCl2 and 2 mM EDTA, pH 7.4 (5M HCl)) and homogenised with an Ultra-Turrax homogeniser (IKA, Staufen, Germany) for 20 s followed by 4 × 4 strokes in a glass-teflon homogeniser. Homogenised tissue was washed in assay buffer and centrifuged at 500 g for 10 min at 4˚C. The supernatant was then harvested and centrifuged at 40,000 g for 15 min at 4˚C with the resulting pellet resuspended in assay buffer and centrifuged a second time at 40,000 g for 15 min at 4˚C. Membrane pellets were then passed 10 × through a 0.22 mm needle, resuspended in assay buffer and protein concentration determined using the bicinchoninic acid method [
All radioligand binding experiments were performed in 96-deep well plates at 37˚C. Binding buffer consisted of RPMI1640 containing 100 µM Gpp(NH)p (pH 7.4). Radioligands ([3H]propranolol or [3H]vilanterol) were incubated with 50 μg/well membranes and either vehicle (1% DMSO to give total radioligand binding) or unlabelled β2-AR agonists/antagonists (10 µM). Non-specific binding (NSB) values were determined by either 10 μM ICI118551 or salmeterol and were used to calculate specific binding. [3H]propranolol saturation binding curve studies were completed in the presence of 0.1 µM of CGP20712 (selective β1-adrenoceptor (β1-AR) antagonist [
β2-AR binding parameters in HLPMs (equilibrium dissociation constant (KD) and total number of receptors (Bmax) were calculated as described under Data Analysis) using 10 µM ICI118551, a selective β2-adrenoceptor (β2-AR) antagonist, to determine NSB. For saturation binding, membranes were incubated with increasing concentrations of [3H]propranolol (~0.04 to 6.0 nM) or [3H]vilanterol (~0.03 - 5.3 nM) for 1 h prior to filtration. Single shot competition displacement studies were also completed where membranes were incubated with a fixed concentration of either [3H]propranolol (~1.7 nM) or [3H] vilanterol (~0.3 nM) and 10 µM of β2-AR unlabelled agonist/antagonist.
Analysis of all radioligand binding experiments was completed using Prism 5.0 (GraphPad Software, San Diego, CA, USA). Specific binding data from saturation experiments were fitted to a one affinity site model to determine KD and Bmax values. Unless otherwise indicated, data shown graphically are mean ± standard error of the mean (SEM). For comparison of model fitting the extra sum-of-squares F test was used with a threshold P < 0.05.
All statistical analyses were completed using SAS® (SAS Institute Inc., NC, USA) and differences of P < 0.05 were considered to be statistically significant. Statistical significance between two data sets was tested using a Student’s unpaired t-test. One-way analysis of variance (ANOVA) was used for comparison of more than two datasets to highlight specific inter-group Pvalues, with Holm’s method [
[3H] vilanterol and [3H] propranolol saturation binding studies were carried out to determine binding affinity and compare the receptor populations labelled in HLPMs. Specific binding data from saturation experiments for both radioligands were best fitted to a one affinity site model (
This analysis resulted in a pKD for [3H]vilanterol of 8.80 ± 0.30 and 8.98 ± 0.14 for [3H]propranolol (n = 6). The average Hill slope coefficient for [3H]vilanterol was 1.17 (0.94, 1.41) and for [3H] propranolol was 0.80 (0.67, 0.93) (n = 6 (2 donors, n = 3/donor), 95% confidence limits shown in parentheses). The Bmax values for [3H] vilanterol and [3H]propranolol saturation binding were 0.37 ± 0.07 and 0.51 ± 0.11 pmol/mg (n = 6 (2 donors, n = 3/donor)) respectively, with no significant difference
observed between radioligands (Student’s t-test, P > 0.05). This suggested that both radioligands were labeling the same population of receptors.
Competition binding with unlabelled β2-AR agonist and antagonists was determined against [3H]vilanterol at single concentrations (10 µM) in HLPMs following a 1 h incubation period at 37˚C at a concentration of radioligand that ensured measurement of β2-AR binding only (i.e. approximately >400-fold lower concentrations shown to engage other endogenous receptors (screened against panel of 7TM receptors and transporters using radioligand binding assays by Eurofins Panlabs Inc. (Bothell, WA, USA), data not shown) including β1/3-adrenoceptors [
Single concentrations (10 µM) of indacaterol, salmeterol and vilanterol resulted in inhibition of [3H]propranolol binding to levels not significantly different from ICI118551 and propranolol (ANOVA, Bonferroni posttest, P > 0.05) (
LABAs have been used for the last 20 years in the treatment of asthma and COPD to provide continuous relief of symptoms via sustained relaxation of the airways and increased airflow into the lungs. The mechanism accounting for the observed extended duration of airway relaxation following LABA inhalation has never fully been elucidated and although a number of hypotheses
have been put forward [7-9] none of these have been categorically proven. In this study [3H]vilanterol has been used as a tool radioligand in unison with [3H]propranolol to investigate the binding of a range of β2-AR agonists (both short and long acting) and antagonists in sub-cellular membrane preparations generated from human lung tissue. The aim being to provide further evidence to either add weight to or rule out the current theories that have been put forward for the duration of action displayed by LABAs.
Historical studies have been completed using membrane preparations from CHO cells recombinantly expressing the human β2-AR allowing characterisation of the affinity and maximal inhibition of binding of unlabelled ligands using [3H]vilanterol [
Membranes generated from human lung parenchyma contain a range of tissue architecture including membranes from cells making up alveoli, blood vessels and small airways. Therefore, it is worth noting that human lung membranes used in this study will contain a range of receptors in addition to the β2-AR, including the β1-AR subtype. To aid in the dissection of β2-AR versus β1-AR subtype binding, a tritiated version of the nonselective β2-AR antagonist propranolol [
The saturation binding data for [3H]vilanterol and [3H]propranolol showed that both radioligands were labelling the same number of β2-AR binding sites in human lung membranes, that would be predicted to be the β2-AR orthosteric binding site in its low affinity state due to the presence of Gpp(NH)p. Subsequent single concentration competition binding for a range of β2-AR agonists and antagonists against [3H]vilanterol showed a subset of LABAs (indacaterol, salmeterol and vilanterol) inhibiting binding to a significantly greater level than other test agents (
Due to the structural similarities between salmeterol and vilanterol i.e. saligenin head with a long hydrophobic tail (
acLogP values calculated by Daylight (Daylight Chemical Information Systems Inc., Laguna Niguel, CA, USA).
level of [3H]vilanterol binding observed in human lung membranes there is a trend observed that the increased lipophilicity of this structural region (hydrophobic tail) contributes to engagement with the tissue binding site. High lipophilicity per se does not result in an interaction with this secondary site as propranolol and ICI118551 did not bind to the tissue site and have either a comparable or greater cLogP than indacaterol, salmeterol and vilanterol (
The novel evidence for a non-β2-AR human lung tissue site that has been generated in this study does not provide any further evidence for confirmation nor invalidation of either the “microkinetic” [
In summary, it has been shown using β2-AR agonist and antagonist tool radioligands that a tissue binding site distinct from the β2-AR is present in parenchyma membranes prepared from human lung tissue. This non-β2-AR binding site appears to be exclusive to a select number of LABAs (indacaterol, salmeterol and vilanterol), with potentially a link between their lipophilicity and the ability to interact with the site. This may provide a further hypothesis for the duration of action exhibited by these drugs, in addition or as an alternative to the “microkinetic” and “exosite” theories, where binding to a tissue site holds these LABAs in the lung for a longer period of time manifesting in a prolonged activation of the β2-AR and subsequent relief of the symptoms of asthma and COPD.
The author would like to acknowledge Mrs Vikki Barrett, Ms Alison Ford, Prof Richard Knowles and the entire scientific and management team at GlaxoSmithKline that contributed to the discovery, characterisation and progression of vilanterol. I would also like to thank the Respiratory TAU Medicinal Chemistry team (especially Dr Pan Procopiou) at GlaxoSmithKline for the synthesis of indacaterol, salbutamol, salmeterol and vilanterol. The author would also like to acknowledge Dr. Alun Bedding of the Quantitative Sciences Division at GlaxoSmithKline for statistical data analysis.