Formoterol Exerts Anti-Cancer Effects Modulating Oxidative Stress and Epithelial-Mesenchymal Transition Processes in Cigarette Smoke Extract Exposed Lung Adenocarcinoma Cells.

Lung cancer frequently affects patients with Chronic Obstructive Pulmonary Disease (COPD). Cigarette smoke (CS) fosters cancer progression by increasing oxidative stress and by modulating epithelial-mesenchymal transition (EMT) processes in cancer cells. Formoterol (FO), a long-acting ß2-agonist widely used for the treatment of COPD, exerts antioxidant activities. This study explored in a lung adenocarcinoma cell line (A549) whether FO counteracted the effects of cigarette smoke extract (CSE) relative to oxidative stress, inflammation, EMT processes, and cell migration and proliferation. A549 was stimulated with CSE and FO, ROS were evaluated by flow-cytometry and by nanostructured electrochemical sensor, EMT markers were evaluated by flow-cytometry and Real-Time PCR, IL-8 was evaluated by ELISA, cell migration was assessed by scratch and phalloidin test, and cell proliferation was assessed by clonogenic assay. CSE significantly increased the production of ROS, IL-8 release, cell migration and proliferation, and SNAIL1 expression but significantly decreased E-cadherin expression. FO reverted all these phenomena in CSE-stimulated A549 cells. The present study provides intriguing evidence that FO may exert anti-cancer effects by reverting oxidative stress, inflammation, and EMT markers induced by CS. These findings must be validated in future clinical studies to support FO as a valuable add-on treatment for lung cancer management.

Rhinovirus C15 Attenuates Relaxation and cAMP Production in Human Airways and Smooth Muscle.

Rhinoviruses (RVs) evoke as many as 85% of acute asthma exacerbations in children and 50% in adults and can induce airway hyperresponsiveness and decrease efficacy of current therapeutics to provide symptom relief. Using human precision-cut lung slices (hPCLSs), primary human air-liquid interface-differentiated airway epithelial cells (HAECs), and human airway smooth muscle (HASM) as preclinical experimental models, we demonstrated that RV-C15 attenuates agonist-induced bronchodilation. Specifically, airway relaxation to formoterol and cholera toxin, but not forskolin (Fsk), was attenuated following hPCLS exposure to RV-C15. In isolated HASM cells, exposure to conditioned media from RV-exposed HAECs decreased cellular relaxation in response to isoproterenol and prostaglandin E2, but not Fsk. Additionally, cAMP generation elicited by formoterol and isoproterenol, but not Fsk, was attenuated following HASM exposure to RV-C15-conditioned HAEC media. HASM exposure to RV-C15-conditioned HAEC media modulated expression of components of relaxation pathways, specifically GNAI1 and GRK2. Strikingly, similar to exposure to intact RV-C15, hPCLS exposed to UV-inactivated RV-C15 showed markedly attenuated airway relaxation in response to formoterol, suggesting that the mechanism(s) of RV-C15-mediated loss of bronchodilation is independent of virus replication pathways. Further studies are warranted to identify soluble factor(s) regulating the epithelial-driven smooth muscle loss of ß2-adrenergic receptor function.

Combinatory in vitro effects of the ß2-agonists salbutamol and formoterol in skeletal muscle cells.

ß2-agonists are used for the treatment of bronchoconstriction, but also abused in doping. Beside an ergogenic activity ß2-agonists may have also anabolic activity. Therefore, we investigated the anabolic activity and associated molecular mechanisms of Salbutamol (SAL) and Formoterol (FOR) alone, as well as in combination in C2C12 myotubes. In differentiated C2C12 cells, dose-dependent effects of SAL and FOR (alone/in combination) on myotube diameter, myosin heavy chain (MHC) protein expression and the mRNA expression of genes involved in hypertrophy were analyzed. ß2-adrenoceptor 2 (ADRB2), androgen receptor (AR) and estrogen receptor (ER) inhibitors, as well as dexamethasone (Dexa) were co-incubated with the ß2-agonists and myotube diameter was determined. SAL and FOR treatment significantly induced hypertrophy and increased MHC expression and the mRNA expression of Igf1, mTOR, PIk3r1 and AMpKa2. In contrast to an ER inhibitor, the ADRB2 and AR inhibitors, as well as Dexa antagonized FOR and SAL induced hypertrophy. Combined treatment with SAL and FOR resulted in significant additive effects on myotube diameter and MHC expression. Future clinical studies are needed to prove this effect in humans and to evaluate this finding with respect to antidoping regulations.

The ß2-adrenergic receptor agonist formoterol restores mitochondrial homeostasis in glucose-induced renal proximal tubule injury through separate integrated pathways.

Mitochondrial dysfunction drives the development and progression of diabetic kidney disease (DKD). Previously, we discovered that the ß2-adrenergic receptor (AR) agonist formoterol regulates mitochondrial dynamics in the hyperglycemic renal proximal tubule. The goal of this study was to identify signaling mechanisms through which formoterol restores the mitochondrial fission/fusion proteins Drp1 and Mfn1. Using primary renal proximal tubule cells (RPTC), the effect of chronic high glucose on RhoA/ROCK1/Drp1 and Raf/MEK1/2/ERK1/2/Mfn1 signaling was determined. In glucose-treated RPTC, RhoA became hyperactive, leading to ROCK1-induced activation of Drp1. Treatment with formoterol and/or pharmacological inhibitors targeting RhoA, ROCK1 and Drp1 blocked RhoA and Drp1 hyperactivity. Inhibiting this pathway also restored maximal mitochondrial respiration. By preventing Gßγ signaling with gallein, we determined that formoterol signals through the Gßγ subunit of the ß2-AR to restore RhoA and Drp1. Furthermore, formoterol restored this pathway by blocking binding of RhoA with the guanine nucleotide exchange factor p114RhoGEF. Formoterol also restored the mitochondrial fusion protein Mfn1 through a second Gßγ-dependent mechanism composed of Raf/MEK1/2/ERK1/2/Mfn1. Glucose-treated RPTC exhibited decreased Mfn1 activity, which was restored with formoterol. Pharmacological inhibition of Gßγ, Raf and MEK1/2 also restored Mfn1 activity. We demonstrate that glucose promotes the interaction between RhoA and p114RhoGEF, leading to increased RhoA and ROCK1-mediated activation of Drp1, and decreases Mfn1 activity through Raf/MEK1/2/ERK1/2. Formoterol restores these pathways and mitochondrial function in response to elevated glucose by activating separate yet integrative pathways that promote mitochondrial biogenesis, decreased fission and increased fusion in RPTC, further supporting its potential as a therapeutic for DKD.

Restricted physical activity after volumetric muscle loss alters whole-body and local muscle metabolism.

Volumetric muscle loss (VML) is the traumatic loss of skeletal muscle, resulting in chronic functional deficits and pathological comorbidities, including altered whole-body metabolic rate and respiratory exchange ratio (RER), despite no change in physical activity in animal models. In other injury models, treatment with ß2 receptor agonists (e.g. formoterol) improves metabolic and skeletal muscle function. We aimed first to examine if restricting physical activity following injury affects metabolic and skeletal muscle function, and second, to enhance the metabolic and contractile function of the muscle remaining following VML injury through treatment with formoterol. Adult male C57Bl/6J mice (n = 32) underwent VML injury to the posterior hindlimb compartment and were randomly assigned to unrestricted or restricted activity and formoterol treatment or no treatment; age-matched injury naïve mice (n = 4) were controls for biochemical analyses. Longitudinal 24 h evaluations of physical activity and whole-body metabolism were conducted following VML. In vivo muscle function was assessed terminally, and muscles were biochemically evaluated for protein expression, mitochondrial enzyme activity and untargeted metabolomics. Restricting activity chronically after VML had the greatest effect on physical activity and RER, reflected in reduced lipid oxidation, although changes were attenuated by formoterol treatment. Formoterol enhanced injured muscle mass, while mitigating functional deficits. These novel findings indicate physical activity restriction may recapitulate following VML clinically, and adjunctive oxidative treatment may create a metabolically beneficial intramuscular environment while enhancing the injured muscle's mass and force-producing capacity. Further investigation is needed to evaluate adjunctive oxidative treatment with rehabilitation, which may augment the muscle's regenerative and functional capacity following VML. KEY POINTS: The natural ability of skeletal muscle to regenerate and recover function is lost following complex traumatic musculoskeletal injury, such as volumetric muscle loss (VML), and physical inactivity following VML may incur additional deleterious consequences for muscle and metabolic health. Modelling VML injury-induced physical activity restriction altered whole-body metabolism, primarily by decreasing lipid oxidation, while preserving local skeletal muscle metabolic activity. The ß2 adrenergic receptor agonist formoterol has shown promise in other severe injury models to improve regeneration, recover function and enhance metabolism. Treatment with formoterol enhanced mass of the injured muscle and whole-body metabolism while mitigating functional deficits resulting from injury. Understanding of chronic effects of the clinically available and FDA-approved pharmaceutical formoterol could be a translational option to support muscle function after VML injury.

Membrane-Facilitated Receptor Access and Binding Mechanisms of Long-Acting ß2-Adrenergic Receptor Agonists.

The drugs salmeterol, formoterol, and salbutamol constitute the frontline treatment of asthma and other chronic pulmonary diseases. These drugs activate the ß2-adrenergic receptors (ß2-AR), a class A G protein-coupled receptor (GPCR), and differ significantly in their clinical onset and duration of actions. According to the microkinetic model, the long duration of action of salmeterol and formoterol compared with salbutamol were attributed, at least in part, to their high lipophilicity and increased local concentrations in the membrane near the receptor. However, the structural and molecular bases of how the lipophilic drugs reach the binding site of the receptor from the surrounding membrane remain unknown. Using a variety of classic and enhanced molecular dynamics simulation techniques, we investigated the membrane partitioning characteristics, binding, and unbinding mechanisms of the ligands. The obtained results offer remarkable insight into the functional role of membrane lipids in the ligand association process. Strikingly, salmeterol entered the binding site from the bilayer through transmembrane helices 1 and 7. The entry was preceded by membrane-facilitated rearrangement and presentation of its phenyl-alkoxy-alkyl tail as a passkey to an access route gated by F193, a residue known to be critical for salmeterol's affinity. Formoterol's access is through the aqueous path shared by other ß2-AR agents. We observed a novel secondary path for salbutamol that is distinct from its primary route. Our study offers a mechanistic description for the membrane-facilitated access and binding of ligands to a membrane protein and establishes a groundwork for recognizing membrane lipids as an integral component in the molecular recognition process. SIGNIFICANCE STATEMENT: The cell membrane's functional role behind the duration of action of long-acting ß2-adrenergic receptor (ß2-AR) agonists such as salmeterol has been a subject of debate for a long time. This study investigated the binding and unbinding mechanisms of the three commonly used ß2-AR agonists, salmeterol, formoterol, and salbutamol, using advanced simulation techniques. The obtained results offer unprecedented insights into the active role of membrane lipids in facilitating access and binding of the ligands, affecting the molecular recognition process and thus their pharmacology.

Regulation of mitochondrial dynamics and energetics in the diabetic renal proximal tubule by the ß2-adrenergic receptor agonist formoterol.

Diabetes is a prevalent metabolic disease that contributes to ∼50% of all end-stage renal disease and has limited treatment options. We previously demonstrated that the ß2-adrenergic receptor agonist formoterol induced mitochondrial biogenesis and promoted recovery from acute kidney injury. Here, we assessed the effects of formoterol on mitochondrial dysfunction and dynamics in renal proximal tubule cells (RPTCs) treated with high glucose and in a mouse model of type 2 diabetes. RPTCs exposed to 17 mM glucose exhibited increased electron transport chain (ETC) complex I, II, III, and V protein levels and reduced ATP levels and uncoupled oxygen consumption rate compared with RPTCs cultured in the absence of glucose or osmotic controls after 96 h. ETC proteins, ATP, and oxygen consumption rate were restored in RPTCs treated with formoterol. RPTCs exposed to high glucose had increased phospho-dynamin-related protein 1 (Drp1), a mitochondrial fission protein, and decreased mitofusin 1 (Mfn1), a mitochondrial fusion protein. Formoterol treatment restored phospho-Drp1 and Mfn1 to control levels. Db/db and nondiabetic (db/m) mice (10 wk old) were treated with formoterol or vehicle for 3 wk and euthanized. Db/db mice showed increased renal cortical ETC protein levels in complexes I, III, and V and decreased ATP; these changes were prevented by formoterol. Phospho-Drp1 was increased and Mfn1 was decreased in db/db mice, and formoterol restored both to control levels. Together, these findings demonstrate that hyperglycemic conditions in vivo and exposure of RPTCs to high glucose similarly alter mitochondrial bioenergetic and dynamics profiles and that treatment with formoterol can reverse these effects. Formoterol may be a promising strategy for treating early stages of diabetic kidney disease.

Molecular basis of ß-arrestin coupling to formoterol-bound ß1-adrenoceptor.

The ß1-adrenoceptor (ß1AR) is a G-protein-coupled receptor (GPCR) that couples1 to the heterotrimeric G protein Gs. G-protein-mediated signalling is terminated by phosphorylation of the C terminus of the receptor by GPCR kinases (GRKs) and by coupling of ß-arrestin 1 (ßarr1, also known as arrestin 2), which displaces Gs and induces signalling through the MAP kinase pathway2. The ability of synthetic agonists to induce signalling preferentially through either G proteins or arrestins-known as biased agonism3-is important in drug development, because the therapeutic effect may arise from only one signalling cascade, whereas the other pathway may mediate undesirable side effects4. To understand the molecular basis for arrestin coupling, here we determined the cryo-electron microscopy structure of the ß1AR-ßarr1 complex in lipid nanodiscs bound to the biased agonist formoterol5, and the crystal structure of formoterol-bound ß1AR coupled to the G-protein-mimetic nanobody6 Nb80. ßarr1 couples to ß1AR in a manner distinct to that7 of Gs coupling to ß2AR-the finger loop of ßarr1 occupies a narrower cleft on the intracellular surface, and is closer to transmembrane helix H7 of the receptor when compared with the C-terminal α5 helix of Gs. The conformation of the finger loop in ßarr1 is different from that adopted by the finger loop of visual arrestin when it couples to rhodopsin8. ß1AR coupled to ßarr1 shows considerable differences in structure compared with ß1AR coupled to Nb80, including an inward movement of extracellular loop 3 and the cytoplasmic ends of H5 and H6. We observe weakened interactions between formoterol and two serine residues in H5 at the orthosteric binding site of ß1AR, and find that formoterol has a lower affinity for the ß1AR-ßarr1 complex than for the ß1AR-Gs complex. The structural differences between these complexes of ß1AR provide a foundation for the design of small molecules that could bias signalling in the ß-adrenoceptors.

Stereoselective cell uptake of adrenergic agonists and antagonists by organic cation transporters.

Stereoselectivity is well described for receptor binding and enzyme catalysis, but so far has only been scarcely investigated in carrier-mediated membrane transport. We thus studied transport kinetics of racemic (anti)adrenergic drugs by the organic cation transporters OCT1 (wild-type and allelic variants), OCT2, OCT3, MATE1, and MATE2-K with a focus on stereospecificity. OCT1 showed stereoselective uptake with up to 2-fold higher vmax over their corresponding counterpart enantiomers for (R,R)-fenoterol, (R,R)-formoterol, (S)-salbutamol, (S)-acebutolol, and (S)-atenolol. Orciprenaline and etilefrine were also transported stereoselectively. The Km was 2.1-fold and 1.5-fold lower for the (S,S)-enantiomers of fenoterol and formoterol, while no significant difference in Km was seen for the other aforementioned drugs. Common OCT1 variants showed similar enantiopreference to wild-type OCT1, with a few notable exceptions (e.g. a switch in enantiospecificity for fenoterol in OCT1*2 compared to the wild-type). Other cation transporters showed strong differences to OCT1 in stereoselectivity and transport activity: The closely related OCT2 displayed a 20-fold higher vmax for (S,S)-fenoterol compared to (R,R)-fenoterol and OCT2 and OCT3 showed 3.5-fold and 4.6-fold higher vmax for the pharmacologically active (R)-salbutamol over (S)-salbutamol. MATE1 and MATE2-K generally mediated transport with a higher capacity but lower affinity compared to OCT1, with moderate stereoselectivity. Our kinetic studies showed that significant stereoselectivity exists in solute carrier-mediated membrane transport of racemic beta-adrenergic drugs with surprising, and in some instances even opposing, preferences between closely related organic cation transporters. This may be relevant for drug therapy, given the strong involvement of these transporters in hepatic and renal drug elimination.

Use of functional respiratory imaging to characterize the effect of inhalation profile and particle size on lung deposition of inhaled corticosteroid/long-acting ß2-agonists delivered via a pressurized metered-dose inhaler.

BACKGROUND: Functional respiratory imaging (FRI) uses three-dimensional models of human lungs and computational fluid dynamics to simulate functional changes within airways and predict the deposition of inhaled drugs. This study used FRI to model the effects of different patient inhalation and drug formulation factors on lung deposition of an inhaled corticosteroid/long-acting ß2-agonist (ICS/LABA) combination, administered by a pressurized metered-dose inhaler. METHODS: Three-dimensional models of the lungs of six patients with asthma (mean forced expiratory volume in 1 s, 83%), treated with an ICS/LABA, were included. FRI modelling was used to simulate (1) the effects on lung deposition of inhalation duration and particle size [fine particle fraction (FPF), proportion of particles <5 µm; and mass median aerodynamic diameter (MMAD), average size of inhalable particles]; (2) deposition of fluticasone propionate/formoterol (FP/FORM) 125/5 µg; and (3) how inhalation profiles and flow rates affected FP/FORM deposition. RESULTS: Total lung depositions (TLDs) following 1-, 3- and 5-s inhalations were 22.8%, 36.1% and 41.6% (metered dose), respectively, and central-to-peripheral deposition (C:P) ratios were 1.81, 0.86 and 0.61, respectively. TLD increased with increasing FPF, from ~8% at 10% FPF to ~36% at 40% FPF (metered dose); by contrast, MMAD had little effect on TLD, which was similar across MMADs (1.5-4.5 µm) at each FPF. FP/FORM deposited throughout central and peripheral airways with gradual (sinusoidal) and sharp (rapid) inhalations. TLD ranged from 35.8 to 44.0% (metered dose) for gradual and sharp inhalations at 30 and 60 L/min mean flow rates. CONCLUSIONS: These data provide important insights into the potential effects of inhalation characteristics (inhalation profile and duration) and aerosol formulation (FPF) on lung deposition of inhaled therapies. FRI thus represents a useful alternative to scintigraphy techniques. Future FRI studies will further our understanding of the deposition of inhaled drugs and help improve the management of asthma.

Beta-2 Adrenergic Agonists Are Substrates and Inhibitors of Human Organic Cation Transporter 1.

Beta-2-adrenergic agonists are first line therapeutics in the treatment of asthma and chronic obstructive pulmonary disease (COPD). Upon inhalation, bronchodilation is achieved after binding to ß2-receptors, which are primarily localized on airway smooth muscle cells. Given that ß2-adrenergic agonists chemically are bases, they carry net positive charge at physiologic pH value in the lungs (i.e., pH 7.4). Here, we studied whether ß2-agonists interact with organic cation transporters (OCT) and whether this interaction exerted an influence on their passage across the respiratory epithelium to their target receptors. [14C]-TEA uptake into proximal (i.e., Calu-3) and distal (i.e., A549 and NCI-H441) lung epithelial cells was significantly reduced in the presence of salbutamol sulfate, formoterol fumarate, and salmeterol xinafoate in vitro. Expression of all five members of the OCT/N family has been confirmed in human pulmonary epithelial cells in situ and in vitro, which makes the identification of the transporter(s) responsible for the ß2-agonist interaction challenging. Thus, additional experiments were carried out in HEK-293 cells transfected with hOCT1-3. The most pronounced inhibition of organic cation uptake by ß2-agonists was observed in hOCT1 overexpressing HEK-293 cells. hOCT3 transfected HEK-293 cells were affected to a lesser extent, and in hOCT2 transfectants only marginal inhibition of organic cation uptake by ß2-agonists was observed. Bidirectional transport studies across confluent NCI-H441 cell monolayers revealed a net absorptive transport of [3H]-salbutamol, which was sensitive to inhibition by the OCT1 modulator, verapamil. Accordingly, salbutamol uptake into hOCT1 overexpressing HEK-293 cells was time- and concentration-dependent and could be completely blocked by decynium-22. Taken together, our data suggest that ß2-agonists are specific substrates and inhibitors of OCT1 in human respiratory epithelial cells and that this transporter might play a role in the pulmonary disposition of drugs of this class.