Cloning and Functional Characterization of Dog OCT1 and OCT2: Another Step in Exploring Species Differences in Organic Cation Transporters.

OCT1 and OCT2 are polyspecific membrane transporters that are involved in hepatic and renal drug clearance in humans and mice. In this study, we cloned dog OCT1 and OCT2 and compared their function to the human and mouse orthologs. We used liver and kidney RNA to clone dog OCT1 and OCT2. The cloned and the publicly available RNA-Seq sequences differed from the annotated exon-intron structure of OCT1 in the dog genome CanFam3.1. An additional exon between exons 2 and 3 was identified and confirmed by sequencing in six additional dog breeds. Next, dog OCT1 and OCT2 were stably overexpressed in HEK293 cells and the transport kinetics of five drugs were analyzed. We observed strong differences in the transport kinetics between dog and human orthologs. Dog OCT1 transported fenoterol with 12.9-fold higher capacity but 14.3-fold lower affinity (higher KM) than human OCT1. Human OCT1 transported ipratropium with 5.2-fold higher capacity but 8.4-fold lower affinity than dog OCT1. Compared to human OCT2, dog OCT2 showed 10-fold lower transport of fenoterol and butylscopolamine. In conclusion, the functional characterization of dog OCT1 and OCT2 reported here may have implications when using dogs as pre-clinical models as well as for drug therapy in dogs.

Acute tocolysis for intrapartum nonreassuring fetal status: how often does it prevent cesarean delivery? A systematic review and meta-analysis of randomized controlled trials.

OBJECTIVE: This study aimed to evaluate the effectiveness of intrapartum acute tocolysis for nonreassuring fetal heart rate tracing in decreasing the incidence of cesarean delivery. Secondary outcomes included modes of delivery other than cesarean delivery, successful acute tocolysis, time-to-delivery interval, and short-term perinatal outcomes. DATA SOURCES: Searches were performed in MEDLINE/PubMed, Embase, Scopus, the Cochrane Central Register of Controlled Trials and Reviews, ClinicalTrials.gov, and the International Clinical Trials Registry Platform from the inception of each database until February 2022. STUDY ELIGIBILITY CRITERIA: Selection criteria included randomized controlled trials of laboring patients with singleton gestations randomized to receive intrapartum acute tocolysis for nonreassuring fetal heart rate tracing, as defined by the original trial. METHODS: All analyses were done using an intention-to-treat approach, evaluating women according to the treatment group to which they were randomly allocated in the original trials. A frequentist network-meta-analysis was performed. RESULTS: Four randomized clinical trials were eligible, including 605 patients with nonreassuring fetal heart rate tracing and singleton gestations at gestational ages >32 weeks. The cesarean delivery rate was similar among patients managed with different types of acute tocolysis. Acute tocolysis, compared with emergency delivery, was associated with improved neonatal acid-base status (notably decreasing the prevalence of base deficit >12 mmol/L [beta-2 agonists odds ratio, 0.61; 95% confidence interval, 0.37-0.99] and the rate of neonatal intensive care unit admission [beta-2 agonists odds ratio, 0.42; 95% confidence interval, 0.22-0.78]) and with an increase in the time-to-delivery interval (beta-2 agonists mean difference, 17.62 minutes; 95% confidence interval, 15.66-19.58); there was no reduction of cesarean delivery rate, showing an increased rate with atosiban and beta-2 agonists. CONCLUSION: The cesarean delivery rate was not reduced by acute tocolysis when used for nonreassuring fetal heart rate tracing during labor. Acute tocolysis is associated with improved short-term fetal outcomes and safely increases the time-to-delivery interval.

Validation of Fenoterol to Study ß2-Adrenoceptor Function in the Rat Urinary Bladder.

Fenoterol is a ß2-adrenoceptor (AR)-selective agonist that is commonly used to investigate relaxation responses mediated by ß2-AR in smooth muscle preparations. Some data have questioned this because fenoterol had low potency in the rat urinary bladder when a muscarinic agonist was used as a pre-contraction agent and because some investigators proposed that fenoterol may act in part via ß3-AR. We designed the present study to investigate whether fenoterol is a proper pharmacological tool to study ß2-AR-mediated relaxation responses in the rat urinary bladder. Firstly, we have compared the effect of pre-contraction agents on fenoterol potency and found that fenoterol potency was about 1.5 log units greater against KCl than carbachol (pEC50 7.19 ± 0.66 and 5.62 ± 1.09 of KCl and of carbachol, respectively). To test the selectivity of fenoterol, we have determined the effects of the ß2-AR antagonist ICI 118,551 and the ß3-AR antagonist L 748,337 on relaxation responses to fenoterol. While 300 nM L 748,337 had little effect on the potency of fenoterol (pEC50 6.56 ± 0.25 and 6.33 ± 0.61 in the absence and presence of L 748,337, respectively), the relaxation curve for fenoterol was right-shifted in the presence 300 nM ICI 118,551 (pEC50 5.03 ± 0.18). Thus, we conclude that fenoterol is a proper pharmacological tool to assess ß2-AR-mediated responses in the rat urinary bladder and most likely in other smooth-muscle preparations containing multiple subtypes of the ß-AR.

Effects of a Common Eight Base Pairs Duplication at the Exon 7-Intron 7 Junction on Splicing, Expression, and Function of OCT1.

Organic cation transporter 1 (OCT1, SLC22A1) is localized in the sinusoidal membrane of human hepatocytes and mediates hepatic uptake of weakly basic or cationic drugs and endogenous compounds. Common amino acid substitutions in OCT1 were associated with altered pharmacokinetics and efficacy of drugs like sumatriptan and fenoterol. Recently, the common splice variant rs35854239 has also been suggested to affect OCT1 function. rs35854239 represents an 8 bp duplication of the donor splice site at the exon 7-intron 7 junction. Here we quantified the extent to which this duplication affects OCT1 splicing and, as a consequence, the expression and the function of OCT1. We used pyrosequencing and deep RNA-sequencing to quantify the effect of rs35854239 on splicing after minigene expression of this variant in HepG2 and Huh7 cells and directly in human liver samples. Further, we analyzed the effects of rs35854239 on OCT1 mRNA expression in total, localization and activity of the resulting OCT1 protein, and on the pharmacokinetics of sumatriptan and fenoterol. The 8 bp duplication caused alternative splicing in 38% (deep RNA-sequencing) to 52% (pyrosequencing) of the minigene transcripts when analyzed in HepG2 and Huh7 cells. The alternatively spliced transcript encodes for a truncated protein that after transient transfection in HEK293 cells was not localized in the plasma membrane and was not able to transport the OCT1 model substrate ASP+. In human liver, however, the alternatively spliced OCT1 transcript was detectable only at very low levels (0.3% in heterozygous and 0.6% in homozygous carriers of the 8 bp duplication, deep RNA-sequencing). The 8 bp duplication was associated with a significant reduction of OCT1 expression in the human liver, but explained only 9% of the general variability in OCT1 expression and was not associated with significant changes in the pharmacokinetics of sumatriptan and fenoterol. Therefore, the rs35854239 variant only partially changes splicing, causing moderate changes in OCT1 expression and may be of only limited therapeutic relevance.

Fenoterol and dobutamine as SARS-CoV-2 main protease inhibitors: A virtual screening study.

Global health is under heavy threat by a worldwide pandemic caused by a new type of coronavirus (COVID-19) since its rapid spread in China in 2019 [1]. Currently, there are no approved specific drugs and effective treatment for COVID-19 infection, but several available drugs are known to facilitate tentative treatment. Since drug design, development and testing procedures are time-consuming [2], [1], [2], [3], virtual screening studies with the aid of available drug databases take the initiative at this point and save the time. Besides, drug repurposing strategies promises to identify new agents for the novel diseases in a time-critical fashion. In this study, we used structure based virtual screening method on FDA approved drugs and compounds in clinical trials. As a result of this study we choose three most prominent compounds for further studies. Here we show that these three compounds (dobutamine and its two derivatives) can be considered as promising inhibitors for SARS-CoV-2 main protease and results also demonstrate the possible interactions of dobutamine and its derivatives with SARS-CoV-2 main protease (6W63) [6]. Our efforts in this work directly address current urgency of a new drug discovery against COVID-19.

Short-acting inhaled ß2-agonists: why, whom, what, how?

We showed the present data about the efficacy and safety of inhaled short-acting ß2-agonists (SABA), such as salbutamol and fenoterol, in the management of obstructive diseases in children and adults. Our work discusses major mechanisms of action, clinical effects, possible side effects and indications of inhaled SABA. We presented current recommendations for the position of SABA in the therapy of obstructive diseases in children and adults, particularly in asthma and chronic obstructive pulmonary disease.

Antenatal exposure to fenoterol is not associated with the development of retinopathy of prematurity in infants born before 32 weeks of gestation.

PURPOSE: Despite safety concerns, ß2-sympathomimetics are still widely used as tocolytic agents. ß-Blockers in turn are used to treat vasculo-proliferative diseases of the newborn such as retinopathy of prematurity (ROP), which may lead to visual impairment and blindness. The scope of this study was to investigate whether antenatal exposure to the ß2-sympathomimetic fenoterol contributes to the development of ROP. METHODS: For this single-center retrospective case-control study of prospectively collected clinical data, all infants born before 32 weeks of gestation between 2001 and 2012 were included. The association of prenatal exposure to fenoterol and the development of ROP were analyzed by multivariate logistic regression. RESULTS: n = 1134 infants < 32 weeks of gestation were screened for eligibility, out of which n = 722 met the inclusion criteria. Exposure to fenoterol (n = 505) was not associated with a higher rate of ROP (OR 0.721, 95% CI 0.463-1.122). Further, duration of exposure (days) did not alter the incidence of ROP (OR 1.001, 95% CI 0.986-1.016). Frequency distribution of different ROP stages and the need for therapeutic intervention was also not affected by prenatal exposure to fenoterol. Risk factors for the development of ROP like low birth weight, low gestational age, prolonged respiratory support and multiple gestation were confirmed in our large study cohort. CONCLUSION: ß2-Sympathomimetic tocolysis does not increase the rate of ROP in premature infants born < 32 weeks of gestation. Our results render fenoterol a safe tocolytic agent regarding neonatal ROP development.

Functions of beta2-adrenergic receptor in human periodontal ligament cells.

Adrenergic receptors (ARs) are receptors of noradrenalin and adrenalin, of which there are nine different subtypes. In particular, ß2 adrenergic receptor (ß2-AR) is known to be related to the restoration and maintenance of homeostasis in bone and cardiac tissues; however, the functional role of signaling through ß2-AR in periodontal ligament (PDL) tissue has not been fully examined. In this report, we investigated that ß2-AR expression in PDL tissues and their features in PDL cells. ß2-AR expressed in rat PDL tissues and human PDL cells (HPDLCs) derived from two different patients (HPDLCs-2G and -3S). Rat PDL tissue with occlusal loading showed high ß2-AR expression, while its expression was downregulated in that without loading. In HPDLCs, ß2-AR expression was increased exposed to stretch loading. The gene expression of PDL-related molecules was investigated in PDL clone cells (2-23 cells) overexpressing ß2-AR. Their gene expression and intracellular cyclic adenosine monophosphate (cAMP) levels were also investigated in HPDLCs treated with a specific ß2-AR agonist, fenoterol (FEN). Overexpression of ß2-AR significantly promoted the gene expression of PDL-related molecules in 2 to 23 cells. FEN led to an upregulation in the expression of PDL-related molecules and increased intracellular cAMP levels in HPDLCs. In both HPDLCs, inhibition of cAMP signaling by using protein kinase A inhibitor suppressed the FEN-induced gene expression of α-smooth muscle actin. Our findings suggest that the occlusal force is important for ß2-AR expression in PDL tissue and ß2-AR is involved in fibroblastic differentiation and collagen synthesis of PDL cells. The signaling through ß2-AR might be important for restoration and homeostasis of PDL tissue.

Relaxing effects of clenbuterol, ritodrine, salbutamol and fenoterol on the contractions of horse isolated bronchi induced by different stimuli.

ß2-adrenoceptor agonists are considered the most effective drugs to counteract bronchoconstriction in horses with asthma, but only clenbuterol is commonly employed in clinical practice. We evaluated the effects of different selective ß2 agonists: clenbuterol, ritodrine, salbutamol, and fenoterol on the contractions of isolated bronchial muscle of horses induced by electrical field stimulation (EFS), carbachol, histamine, and KCl. All ß2 agonists reduced the amplitude of contraction induced by the different stimuli but with variable efficacy and potency. Fenoterol and salbutamol were more effective than clenbuterol in relaxing the bronchial contractions induced by EFS and histamine, and were able to completely abolish carbachol-induced contractions, unlike clenbuterol and ritodrine. The respective potency values (pEC50) of clenbuterol, ritodrine, salbutamol, and fenoterol were 7.74 ±â€¯0.20, 7.77 ±â€¯0.17, 7.30 ±â€¯0.23, 8.01 ±â€¯0.13, for EFS-induced contractions; 8.39 ±â€¯0.26, 5.49 ±â€¯0.28, 6.63 ±â€¯0.14, 7.68 ±â€¯0.11, for carbachol-induced contraction; 7.39 ±â€¯0.27, 7.04 ±â€¯0.28, 6.45 ±â€¯0.34, 7.34 ±â€¯0.22, for histamine-induced contraction; 7.15 ±â€¯0.06, 6.07 ±â€¯0.20, 6.48 ±â€¯0.14, 6.70 ±â€¯0.18, for KCl-induced contraction. Salbutamol and fenoterol showed a higher efficacy than clenbuterol in relaxing horse bronchial muscle pre-contracted by most stimuli. Clenbuterol displayed a good potency but a rather low efficacy, and this may be due to its partial agonist nature; ritodrine showed lower or not significantly different efficacy and potency compared to the other agonists. An evaluation of the clinical efficacy by fenoterol and salbutamol in horses with asthma could be of great interest to assess if they could represent more effective bronchodilators compared to clenbuterol.

Utility of Europium ion characteristic peak for quantitation of Fenoterol hydrobromide and Salmeterol xinafoate in different matrices; application to stability studies.

A simple selective luminescent dependent approach was established for quantitation of two selective ß2 agonists namely; Fenoterol hydrobromide (FEN) and Salmeterol xinafoate (SAL). This approach utilizes the capability of the cited drugs to undergo a complexation reaction with Europium ion (Eu3+) in the presence of 1,10-phenanthroline as a co-ligand. The resultant complex leads to a hypersensitive transition and enhancement of the Eu3+ emission peak at 615nm (279nm excitation). Under the optimized conditions, the rectilinear concentration plots of both drugs were (70-1500ngmL-1) and (100-2000ngmL-1) with limit of quantitation 51.3 and 84.4ngmL-1 for FEN and SAL, respectively. The luminescence properties of the complex and its optimum formation conditions were carefully investigated according to the regulations of ICH and the method was successfully applied in plasma. The good accuracy and selectivity of the suggested method allowed extending the proposed protocol into stability study of the cited drugs.

Effect of betamethasone, indomethacin and fenoterol on neonatal and maternal mononuclear cells stimulated with Escherichia coli.

Despite considerable progress in the field of perinatal care, infectious diseases, especially when caused by gram negative bacteria, remain a major reason for neonatal morbidity and mortality. Notably infants born prematurely and those with very low birth weight are at risk due to their immature and deficient immune system and their prolonged hospitalization which promotes nosocomial infections. In case of impending preterm birth, betamethasone is given to induce lung maturation and tocolytic agents like indomethacin or fenoterol are administered to suppress premature labor. The aim of this study was to analyze the effects of these drugs on the immune system of mothers and neonates. Therefore, mononuclear cells from cord blood and peripheral maternal blood were stimulated with Escherichia coli and incubated with betamethasone, indomethacin and fenoterol. Subsequently the effect of the treatment on cytokine production was determined. Betamethasone alone and in combination with tocolytic agents inhibited the production of pro- and anti-inflammatory cytokines. Not only does betamethasone dampen the immune response by reducing the production of cytokines, it also has a variety of other detrimental short- and long-term effects on the neonate. In conclusion we would recommend using biological markers to determine if premature labor actually leads to preterm birth and subsequently administer betamethasone only to mothers giving birth prematurely.

Discovery of ß-arrestin-biased ß2-adrenoceptor agonists from 2-amino-2-phenylethanol derivatives.

ß-Arrestins are a small family of proteins important for signal transduction at G protein-coupled receptors (GPCRs). ß-Arrestins are involved in the desensitization of GPCRs. Recently, biased ligands possessing different efficacies in activating the G protein- versus the ß-arrestin-dependent signals downstream of a single GPCR have emerged, which can be used to selectively modulate GPCR signal transduction in such a way that desirable signals are enhanced to produce therapeutic effects while undesirable signals of the same GPCR are suppressed to avoid side effects. In the present study, we evaluated agonist bias for compounds developed along a drug discovery project of ß2-adrenoceptor agonists. About 150 compounds, including derivatives of fenoterol, 2-amino-1-phenylethanol and 2-amino-2-phenylethanol, were obtained or synthesized, and initially screened for their ß-adrenoceptor-mediated activities in the guinea pig tracheal smooth muscle relaxation assay or the cardiomyocyte contractility assay. Nineteen bioactive compounds were further assessed using both the HTRF cAMP assay and the PathHunter ß-arrestin assay. Their concentration-response data in stimulating cAMP synthesis and ß-arrestin recruitment were applied to the Black-Leff operational model for ligand bias quantitation. As a result, three compounds (L-2, L-4, and L-12) with the core structure of 5-(1-amino-2-hydroxyethyl)-8-hydroxyquinolin-2(1H)-one were identified as a new series of ß-arrestin-biased ß2-adrenoceptor agonists, whereas salmeterol was found to be Gs-biased. These findings would facilitate the development of novel drugs for the treatment of both heart failure and asthma.

Atosiban efficacy and safety in pregnant women with threatened preterm delivery: systematic review of the literature with network meta-analysis / Eficacia y seguridad de atosiban en mujeres gestantes con diagnóstico de amenaza de parto pretérmino: revisión sistemática de la literatura con metaanálisis en red

ABSTRACT Objective: To assess the efficacy and safety of atosiban in pregnant women with risk of preterm delivery as compared to nifedipine, indomethacin, terbutaline, fenoterol and placebo. Materials and methods: A systematic literature review was carried out in eight electronic databases, including Medline, Central, and Embase, using free and standardized search terms. Outcomes assessment included time delay until delivery, neonatal mortality, ratio of adverse maternal events, and ratio of neonatal complications. The quality of the evidence was evaluated per study and for the body of evidence and, whenever feasible, the information was synthesized into a meta-analysis. Alternatively, a narrative summary was presented. Results: Eleven studies were included. Atosiban did not show any statistically significant differences in terms of delaying delivery versus other uterine contraction inhibitors. The neonatal mortality was lower compared to indomethacin (RR = 0.21; 95% CI: 0.05 to 0.92), and the percentage of total maternal adverse events was lower compared to fenoterol (RR = 0.16; 95% CI: 0.08 to 0.31), nifedipine (RR = 0.48; 95% CI: 0.3 to 0.78), and terbutaline (RR = 0.44; 95% CI: 0.28 to 0.71). Conclusions: Atosiban has similar efficacy for delivery delay in patients with risk of preterm delivery as compared to other agents (moderate certainty), showing some advantages regarding neonatal mortality (low certainty) versus indomethacin, and compared to fenoterol, nifedipine and terbutaline in terms of maternal adverse events (moderate certainty).

RESUMEN Objetivo: evaluar la eficacia y seguridad de atosiban en gestantes con amenaza de parto pretérmino comparado con nifedipino, indometacina, terbutalina, fenoterol y placebo. Materiales y métodos: se realizó una revisión sistemática de la literatura en ocho bases de datos electrónicas (Medline, Central, Embase, entre otras), mediante términos de búsqueda libres y estandarizados. Los desenlaces evaluados incluyeron tiempo de retardo del parto, mortalidad neonatal, proporción de eventos adversos maternos y proporción de complicaciones neonatales. Se evaluó la calidad de la evidencia por estudio y para el cuerpo de evidencia, y se sintetizó la información mediante metaanálisis, cuando fue posible; de lo contrario, se resumió de forma narrativa. Resultados: se incluyeron once estudios. Atosiban no mostró diferencias estadísticamente significativas en retardo del parto contra otros uteroinhibidores. Mostró menor mortalidad neonatal que la indometacina (RR = 0,21; IC 95 %: 0,05 a 0,92), y menor proporción de eventos adversos maternos totales que el fenoterol (RR = 0,16; IC 95 %: 0,08 a 0,31), el nifedipino (RR = 0,48; IC 95 %: 0,3 a 0,78) y la terbutalina (RR = 0,44; IC 95 %: 0,28 a 0,71). Conclusiones: atosiban tiene una eficacia similar para retardar el parto ante la amenaza de un parto pretérmino con otros comparadores (certeza moderada), con ventajas frente a indometacina en mortalidad neonatal (certeza baja) y frente a fenoterol, nifedipino y terbutalina en eventos adversos maternos (certeza moderada).

New enhanced spectrofluorimetric approach for picogram detection of Fenoterol hydrobromide through Von Pechman synthesis of coumarins.

A new, specific, precise and very sensitive spectrofluorimetric methodology has been established and approved for determination of Fenoterol hydrobromide (FEN) in its pharmaceutical forms and spiked plasma. The strategy utilized the phenolic nature of FEN and its capacity to undergo Von Pechman synthesis of coumarin. In this study, Fenoterol hydrobromide reacts with ethyl acetoacetate in presence of concentrated sulfuric acid to form an extremely fluorescent coumarin derivative measured at 480 nm (λex: 420 nm). Different reaction variables affecting development and stability of the formed coumarin derivative were precisely examined and enhanced to guarantee greatest sensitivity of the strategy. The recommended procedure was found to obey Beer's law in concentration range of (300-2000) pg mL-1 with quantitation limit 130 pg mL-1, revealing high sensitivity of the suggested method. The proposed procedure was completely examined and approved through the ICH guidelines and was efficiently applied for the determination of the cited drug in spiked plasma and its dosage forms.

Tocolysis with the ß2-sympathomimetic fenoterol does not increase the occurrence of infantile hemangioma in preterm and term infants.

PURPOSE: ß2-sympathomimetics are used in obstetrics as tocolytic agents, despite a remarkable profile of side effects. Recently, the ß2-sympathomimetic tocolytic drug hexoprenaline was identified as an independent risk factor for the development of infantile hemangioma (IH) in preterm infants. The aim of this study was to evaluate whether this observed effect was applicable to other ß2-mimetic tocolytic agents like fenoterol. METHODS: Clinical prospectively collected data of all infants born between 2001 and 2012 and admitted to the neonatal intensive care unit (NICU) at Heidelberg University Hospital and respective maternal data were merged. For the current retrospective cohort study, cases (IH) were matched to controls (no IH) at a ratio of 1:4, adjusting for birth weight, gestational age, gender and multiple gestations. Prenatal exposure to fenoterol and perinatal outcome were analyzed in the total cohort and in subgroups. RESULTS: N = 5070 infants were admitted to our neonatal department, out of which n = 172 infants with IH were identified and compared to n = 596 matched controls. Exposure to fenoterol was not associated with a higher rate of IH in the total matched population (OR 0.926, 95% CI 0.619-1.384) or in a subgroup of neonates < 32 weeks of gestation or with a birth weight < 1500 g (OR 1.127, 95% CI 0.709-1.791). In the total matched population, prenatal exposure to glucocorticoids was associated with a reduced occurrence of IH (OR 0.566, 95% CI 0.332-0.964) and neonates with IH showed a prolonged total hospital stay compared to controls (69 vs. 57 days, p = 0.0033). Known risk factors for IH were confirmed by our large study cohort and included female gender, low birth weight, preterm birth and multiple gestations (all p < 0.005). CONCLUSIONS: Exposure to fenoterol during pregnancy does not increase the occurrence of IH. Further studies are needed to explore differences in the risk profiles of different ß2-sympathomimetic tocolytic drugs.

Tensor algebra-based geometric methodology to codify central chirality on organic molecules.

A novel mathematical procedure to codify chiral features of organic molecules in the QuBiLS-MIDAS framework is introduced. This procedure constitutes a generalization to that commonly used to date, where the values 1 and -1 (correction factor) are employed to weight the molecular vectors when each atom is labelled as R (rectus) or S (sinister) according to the Cahn-Ingold-Prelog rules. Therefore, values in the range [Formula: see text] with steps equal to 0.25 may be accounted for. The atoms labelled R or S can have negative and positive values assigned (e.g. -3 for an R atom and 1 for an S atom, or vice versa), opposed values (e.g. -3 for an R atom and 3 for an S atom, or vice versa), positive values (e.g. 3 for an R atom and 1 for an S atom) or negative values (e.g. -3 for an R atom and -1 for an S atom). These proposed Chiral QuBiLS-MIDAS 3D-MDs are real numbers, non-symmetric and reduced to 'classical' (non-chiral) QuBiLS-MIDAS 3D-MDs when symmetry is not codified (correction factor equal to zero). In this report, only the factors with opposed values were considered with the purpose of demonstrating the feasibility of this proposal. From QSAR modelling carried out on four chemical datasets (Cramer's steroids, fenoterol stereoisomer derivatives, N-alkylated 3-(3-hydroxyphenyl)-piperidines, and perindoprilat stereoisomers), it was demonstrated that the use of several correction factors contributes to the building of models with greater robustness and predictive ability than those reported in the literature, as well as with respect to the models exclusively developed with QuBiLS-MIDAS 3D-MDs based on the factor 1 | -1. In conclusion, it can be stated that this novel strategy constitutes a suitable alternative to computed chirality-based descriptors, contributing to the development of good models to predict properties depending on symmetry.

Concurrent activation of ß2-adrenergic receptor and blockage of GPR55 disrupts pro-oncogenic signaling in glioma cells.

Activation of ß2-adrenergic receptor (ß2AR) and deorphanized GPR55 has been shown to modulate cancer growth in diverse tumor types in vitro and in xenograft models in vivo. (R,R')-4'-methoxy-1-naphthylfenoterol [(R,R')-MNF] is a bivalent compound that agonizes ß2AR but inhibits GPR55-mediated pro-oncogenic responses. Here, we investigated the molecular mechanisms underlying the anti-tumorigenic effects of concurrent ß2AR activation and GPR55 blockade in C6 glioma cells using (R,R')-MNF as a marker ligand. Our data show that (R,R')-MNF elicited G1-phase cell cycle arrest and apoptosis, reduced serum-inducible cell motility, promoted the phosphorylation of PKA target proteins, and inhibited constitutive activation of ERK and AKT in the low nanomolar range, whereas high nanomolar levels of (R,R')-MNF were required to block GPR55-mediated cell motility. siRNA knockdown and pharmacological inhibition of ß2AR activity were accompanied by significant upregulation of AKT and ERK phosphorylation, and selective alteration in (R,R')-MNF responsiveness. The effects of agonist stimulation of GPR55 on various readouts, including cell motility assays, were suppressed by (R,R')-MNF. Lastly, a significant increase in phosphorylation-mediated inactivation of ß-catenin occurred with (R,R')-MNF, and we provided new evidence of (R,R')-MNF-mediated inhibition of oncogenic ß-catenin signaling in a C6 xenograft tumor model. Thus, simultaneous activation of ß2AR and blockade of GPR55 may represent a novel therapeutic approach to combat the progression of glioblastoma cancer.

Effects of fenoterol on the skeletal system depend on the androgen level.

BACKGROUND: The role of sympathetic nervous system in the osseous tissue remodeling is not clear enough. METHODS: The effects of fenoterol, a selective ß2-adrenomimetic drug, on the skeletal system of normal and androgen deficient (orchidectomized) rats were studied in vivo. Osteoclastogenesis and mRNA expression in osteoblasts were investigated in vitro in mouse cell cultures. RESULTS: Fenoterol administered to animals with physiological androgen level unfavorably affected the skeletal system, damaging the bone microarchitecture. Androgen deficiency induced osteoporotic changes, and fenoterol protected the osseous tissue from consequences of androgen deficiency. The results of in vitro studies correlated with the in vivo observations. A significantly increased number of osteoclasts in bone marrow cell cultures to which testosterone and fenoterol were added simultaneously was demonstrated. In cultures without the addition of testosterone, fenoterol significantly inhibited osteoclastogenesis in comparison with control cultures. CONCLUSIONS: The results indicate the favorable action of fenoterol in conditions of testosterone deficiency, and its destructive influence upon the skeleton in the presence of androgens. The results confirm the key role of sympathetic nervous system in the regulation of bone remodeling.

Cholesterol regulates contractility and inotropic response to ß2-adrenoceptor agonist in the mouse atria: Involvement of Gi-protein-Akt-NO-pathway.

Majority of cardiac ß2-adrenoceptors is located in cholesterol-rich microdomains. Here, we have investigated the underlying mechanisms by which a slight to moderate cholesterol depletion with methyl-ß-cyclodextrin (MßCD, 1 and 5mM) interferes with contractility and inotropic effect of ß2-adrenergic agonist (fenoterol, 50µM) in the mouse atria. Treatment with MßCD itself increased amplitude of Ca2+ transient but did not change the contraction amplitude due to a clamping action of elevated NO. Cholesterol depletion significantly attenuated the positive inotropic response to fenoterol which is accompanied by increase in NO generation and decrease in Ca2+ transient. Influence of 1mM MßCD on the fenoterol-driven changes in both contractility and NO level was strongly attenuated by inhibition of Gi-protein (pertussis toxin), Akt (Akt 1/2 kinase inhibitor) or NO-synthase (L-NAME). After exposure to 5mM MßCD, pertussis toxin or Akt inhibitor could recover the ß2-agonist effects on contractility, NO production and Ca2+ transient, while L-NAME only reduced NO level. An adenylyl cyclase activator (forskolin, 50nM) had no influence on the MßCD-induced changes in the ß2-agonist effects. Obtained results suggest that slight cholesterol depletion upregulates Gi-protein/Akt/NO-synthase signaling that attenuates the positive inotropic response to ß2-adrenergic stimulation without altering the Ca2+ transient. Whilst moderate cholesterol depletion additionally could suppress the enhancement of the Ca2+ transient amplitude caused by the ß2-adrenergic agonist administration in Gi-protein/Akt-dependent but NO-independent manner.

Anti-inflammatory activities of fenoterol through ß-arrestin-2 and inhibition of AMPK and NF-κB activation in AICAR-induced THP-1 cells.

The AMP-activated protein kinase (AMPK) pathway has been shown to be able to regulate inflammation in several cell lines. We reported that fenoterol, a ß2-adrenergic receptor (ß2-AR) agonist, inhibited lipopolysaccharide (LPS)-induced AMPK activation and inflammatory cytokine production in THP-1 cells, a monocytic cell line in previous studies. 5-amino-1-ß-d-ribofuranosyl-imidazole-4-carboxamide (AICAR) is an agonist of AMPK. Whether AICAR induced AMPK activation and inflammatory cytokine production in THP-1 cells can be inhibited by fenoterol is unknown. In this study, we explored the mechanism of ß2-AR stimulation with fenoterol in AICAR-induced inflammatory cytokine secretion in THP-1 cells. We studied AMPK activation using p-AMPK and AMPK antibodies, nuclear factor-kappa B (NF-κB) activation and inflammatory cytokine secretion in THP-1 cells stimulated by ß2-AR in the presence or absence of AICAR and small interfering RNA (siRNA)-mediated knockdown of ß-arrestin-2 or AMPKα1 subunit. AICAR-induced AMPK activation, NF-κB activation and tumor necrosis factor (TNF)-α release were reduced by fenoterol. In addition, siRNA-mediated knockdown of ß-arrestin-2 abolished fenoterol's inhibition of AICAR-induced AMPK activation and TNF-α release, thus ß-arrestin-2 mediated the anti-inflammatory effects of fenoterol in AICAR-treated THP-1 cells. Furthermore, siRNA-mediated knockdown of AMPKα1 significantly attenuated AICAR-induced NF-κB activation and TNF-α release, so AMPKα1 was a key signaling molecule involved in AICAR-induced inflammatory cytokine production. These data suggested that fenoterol inhibited AICAR-induced AMPK activation and TNF-α release through ß-arrestin-2 in THP-1 cells. Management especially inhibition of AMPK signaling may provide new approaches and strategies for the treatments of immune diseases including inflammatory diseases and other critical illness.