The ß2-adrenergic receptor agonist terbutaline upregulates T helper-17 cells in a protein kinase A-dependent manner.

BACKGROUND: T helper 17 (Th17) cells produce IL-17A cytokine and can exacerbate autoimmune diseases and asthma. The ß2 adrenergic receptor is a g protein-coupled receptor that induces cAMP second messenger pathways. We tested the hypothesis that terbutaline, a ß2-adrenergic receptor-specific agonist, promotes IL-17 secretion by memory Th17 cells in a cAMP and PKA-dependent manner. METHODS: Venous peripheral blood mononuclear cells (PBMC) from healthy human participants were activated with anti-CD3 and anti-CD28 antibodies. Secreted IL-17A was measured by enzyme linked immunosorbent assay, intracellular IL-17A, and RORγ were measured using flow cytometry, and RORC by qPCR. Memory CD3+CD4+CD45RA-CD45RO+ T cells were obtained by immunomagnetic negative selection and activated with tri-antibody complex CD3/CD28/CD2. Secreted IL-17A, intracellular IL-17A, RORC were measured, and phosphorylated-serine133-CREB was measured by western blotting memory Th cells. RESULTS: Terbutaline increased IL-17A (p < 0.001), IL-17A+ cells (p < 0.05), and RORC in activated PBMC and memory Th cells. The PKA inhibitors H89 (p < 0.001) and Rp-cAMP (p < 0.01) abrogated the effects of terbutaline on IL-17A secretion in PBMC and memory T cells. Rolipram increased IL-17A (p < 0.01) to a similar extent as terbutaline. P-Ser133-CREB was increased by terbutaline (p < 0.05) in memory T cells. CONCLUSION: Terbutaline augments memory Th17 cells in lymphocytes from healthy participants. This could exacerbate autoimmune diseases or asthma, in cases where Th17 cells are considered to be pro-inflammatory.

Terbutaline attenuates LPS-induced injury of pulmonary microvascular endothelial cells by cAMP/Epac signaling.

Acute lung injury (ALI), characterized by an acute onset of severe hypoxemia, is a common and devastating syndrome usually triggered by lipopolysaccharide (LPS) infection from bacteria. This study is intended to explore whether terbutaline can alleviate LPS-induced human pulmonary microvascular endothelial cell (HPMVEC) injury through cAMP/Epac signaling. LPS was utilized to induce ALI in HPMVECs, and after exposure of LPS-induced HPMVECs to terbutaline, the cellular functions including cell viability and apoptosis were measured by cell counting kit-8 and terminal deoxynucleotidyl transferase dUTP nick-end labeling. The protein expression related to cAMP/Epac signaling, apoptosis, and that of tight junction and inflammatory factors were evaluated at the same time. The effects of terbutaline on cellular functions were confirmed again after the addition of antagonists of cAMP and Epac, respectively. The levels of both cAMP and Epac reduced by LPS was concentration-dependently increased by terbutaline. The apoptosis and endothelial cell permeability damage of LPS-induced HPMVECs were enhanced after the addition of KT-5720 and ESI-09. The beneficial effects of terbutaline on alleviating the inflammation and apoptosis in HPMVECs injured by LPS are mediated by cAMP/Epac signaling, and this evidence would demonstrate the potential of terbutaline in the treatment of ALI.

G protein-coupled receptor-in-paper, a versatile chromatographic platform to study receptor-drug interaction.

High-performance affinity chromatography is limited by its high cost and high pressure. Paper is made up of porous fiber networks and has the properties of low cost, ease of fabrication, and biodegradable. Due to these advantages, herein, we immobilized beta2-adrenoceptor (ß2-AR) onto the surface of the polytetrafluoroethylene membrane, a paper-based material, and constructed a G protein-coupled receptor (GPCR)-in-paper chromatographic platform. This platform was characterized by Fourier transform infrared spectroscopy, fluorescence analysis, X-ray photoelectron spectroscopy, and chromatographic studies. These morphological and elemental analysis showed that ß2-AR was successfully immobilized on the paper surface. The specific drugs have good retentions on the GPCR-in-paper chromatographic platform. The association constants of salbutamol, terbutaline and bambuterol to ß2-AR were calculated to be 2.02 × 104 M-1, 1.15 × 104 M-1, 1.75 × 104 M-1 by adsorption energy distribution, which were in good line with the values from frontal analysis, zonal elution and previous literatures. We demonstrated that the GPCR-in-paper platform was cost-effective, easy to be modified for protein immobilization, and applicable in the receptor-drug interaction analysis. We believe such a platform sheds new light on paper chromatography for receptor-drug interaction analysis and other applications.

ß2 Adrenergic Regulation of the Phagocytic and Microbicide Capacity of Circulating Monocytes: Influence of Obesity and Exercise.

Obese individuals present anomalous immune/inflammatory responses with dysregulations in neuroendocrine responses and immune/stress feedback mechanisms. In this context, exercise and ß2 adrenergic activation present monocyte-mediated anti-inflammatory effects that are modulated by obesity. However, these anti-inflammatory effects could immunocompromise the monocyte-mediated innate response against a pathogen challenge. Thus, the objective of this work was to evaluate the effect of obesity, and exercise in this condition, on the ß2 adrenergic regulation of the phagocytic and microbicide capacity of circulating monocytes. C57BL/6J mice were allocated to different sedentary or exercised, lean or obese groups. Obese mice showed a lower monocyte-mediated innate response than that of lean mice. Globally, selective ß2 adrenergic receptor agonist terbutaline decreased the innate response of monocytes from lean and obese sedentary animals, whereas exercise stimulated it. Exercise modulates ß2 adrenergic regulation of the innate response in lean and obese animals, with a global stimulatory or neutral effect, thus abolishing the inhibitory effect of terbutaline occurring in sedentary animals. These effects cannot be explained only by changes in the surface expression of toll-like receptors. Therefore, in general, terbutaline does not hinder the effects of regular exercise, but regular exercise does abolish the effects of terbutaline in sedentary individuals.

Interactions of Paraoxonase-1 with Pharmacologically Relevant Carbamates.

Mammalian paraoxonase-1 hydrolyses a very broad spectrum of esters such as certain drugs and xenobiotics. The aim of this study was to determine whether carbamates influence the activity of recombinant PON1 (rePON1). Carbamates were selected having a variety of applications: bambuterol and physostigmine are drugs, carbofuran is used as a pesticide, while Ro 02-0683 is diagnostic reagent. All the selected carbamates reduced the arylesterase activity of rePON1 towards the substrate S-phenyl thioacetate (PTA). Inhibition dissociation constants (Ki), evaluated by both discontinuous and continuous inhibition measurements (progress curves), were similar and in the mM range. The rePON1 displayed almost the same values of Ki constants for Ro 02-0683 and physostigmine while, for carbofuran and bambuterol, the values were approximately ten times lower and two times higher, respectively. The affinity of rePON1 towards the tested carbamates was about 3-40 times lower than that of PTA. Molecular modelling of rePON1-carbamate complexes suggested non-covalent interactions with residues of the rePON1 active site that could lead to competitive inhibition of its arylesterase activity. In conclusion, carbamates can reduce the level of PON1 activity, which should be kept in mind, especially in medical conditions characterized by reduced PON1 levels.

Attenuated Total Reflectance Fourier Transformation Infrared spectroscopy fingerprinted online monitoring of the kinetics of circulating Butyrylcholinesterase enzyme during metabolism of bambuterol.

We have described a continuous flow ATR-FTIR method for measuring some of the Butyrylcholinesterase enzyme kinetics (Km and Vmax). This is done by developing a circulating system to be close as much as possible to the human circulation using human serum as a source of the enzyme with adjusted pH, isotonicity and temperature to give the maximum affinity of the enzyme towards its substrate (bambuterol). The experiment was running continuously for 90 min to monitor the production of terbutaline from the zero time of its appearance with a measured spectrum in each minute using ZnSe prism. The method was selective and successful for determination of Vmax to be 8.16 × 10-8 mol/min/ml and Km to be 2.28 × 10-5 mol, showing high affinity of the enzyme towards its prodrug substrate Bambuterol. This study critically probes the quantitative ability of the ATR-FTIR method for terbutaline, which was validated according to ICH guidelines showing high accuracy 100.39% and high selectivity towards the produced terbutaline, as the produced spectrums considered as fingerprint of each compound.

In vivo metabolism study of (R)-bambuterol in humans using ultra high performance liquid chromatography with tandem mass spectrometry.

(R)-Bambuterol, a selective ß2-adrenoceptor agonist, has been approved as a new drug for the treatment of asthma and chronic obstructive pulmonary disease by the China Food and Drug Administration and is currently under phase I clinical trials. In this study, a combined method based on ultra high performance liquid chromatography with triple quadrupole mass spectrometry and ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry was employed for the identification of the major metabolites of (R)-bambuterol in human plasma and urine after an oral dose of 10 mg. The metabolites were separated by gradient elution program and different sample preparation methods were compared. Totally, 12 metabolites of (R)-bambuterol were identified, including four metabolites in plasma and all 12 metabolites in urine. Among these, four metabolites are reported for the first time. The possible metabolic pathways of (R)-bambuterol were subsequently proposed. The results indicated that (R)-bambuterol was metabolized via hydrolysis, demethylation, oxygenation, glucuronidation, and sulfation pathways in vivo. This study revealed that this combined method was accurate and sensitive to identify the possible metabolites and to better understand the metabolism of (R)-bambuterol in vivo.

Binding studies of terbutaline sulfate to calf thymus DNA using multispectroscopic and molecular docking techniques.

The interaction of terbutaline sulfate (TS) with calf thymus DNA (ctDNA) were investigated by fluorescence quenching, UV-vis absorption, viscosity measurements, ionic strength effect, DNA melting experiments and molecular docking. The binding constants (Ka) of TS to ctDNA were determined as 4.92×10(4), 1.26×10(4) and 1.16×10(4) L mol(-1) at 17, 27 and 37 °C, respectively. Stern-Volmer plots suggested that the quenching of fluorescence of TS by ctDNA was a static quenching. The absorption spectra of TS with ctDNA revealed a slight blue shift and hyperchromic effect. The relative viscosity ctDNA was hardly changed by TS, and melting temperature varied slightly. For the system of TS-ctDNA, the intensity of fluorescence decreased with the increase of ionic strength. Also, the Ka for TS-double stranded DNA (dsDNA) was clearly weaker than that for TS-single stranded DNA (ssDNA). All these results revealed that the binding mode of TS with ctDNA should be groove binding. The enthalpy change and entropy change suggested that van der Waals force or hydrogen bonds was a main binding force between TS and ctDNA. Furthermore, the quantum yield of TS was measured by comparing with the standard solution. Based on the Förster energy transference theory (FRET), the binding distance between the acceptor and donor was calculated. Molecular docking showed that TS was a minor groove binder of ctDNA and preferentially bound to A-T rich regions.

In Vitro Assessment of Uptake and Lysosomal Sequestration of Respiratory Drugs in Alveolar Macrophage Cell Line NR8383.

PURPOSE: To assess accumulation and lysosomal sequestration of 9 drugs used in respiratory indications (plus imipramine as positive control) in the alveolar macrophage (AM) cell line NR8383. METHODS: For all drugs, uptake at 5 µM was investigated at 37 and 4°C to delineate active uptake and passive diffusion processes. Accumulation of basic clarithromycin, formoterol and imipramine was also assessed over 0.1-100 µM concentration range. Lysosomal sequestration was investigated using ammonium chloride (NH4Cl), monensin and nigericin. Impact of lysosomal sequestration on clarithromycin accumulation kinetics was investigated. RESULTS: Both cell-to-medium concentration ratio (Kp) and uptake clearance (CLuptake) ranged > 400-fold for the drugs investigated. The greatest Kp was observed for imipramine (391) and clarithromycin (82), in contrast to no accumulation seen for terbutaline. A concentration-dependent accumulation was evident for the basic drugs investigated. Imipramine and clarithromycin Kp and CLuptake were reduced by 59-85% in the presence of NH4Cl and monensin/nigericin, indicating lysosomal accumulation, whereas lysosomal sequestration was not pronounced for the other 8 respiratory drugs. Clarithromycin uptake rate was altered by NH4Cl, highlighting the impact of subcellular distribution on accumulation kinetics. CONCLUSIONS: This study provides novel evidence of the utility of NR8383 for investigating accumulation and lysosomal sequestration of respiratory drugs in AMs.

Stereoselective inhibition of human butyrylcholinesterase by the enantiomers of bambuterol and their intermediates.

This work describes the sequential hydrolysis of bambuterol enantiomers and their monocarbamate metabolites (MONO) catalyzed by human butyrylcholinesterase (BChE) as well as the enzyme inhibition resulting from this process. Particular emphasis is given to the contribution given by MONO to the enzyme inhibition because it was not fully characterized in previous works. Bambuterol and MONO enantiomers displayed the same time- and concentration-dependent mechanism of interaction with the enzyme. The hydrolysis kinetics of both bambuterol and MONO was enantioselective, and the (R)-enantiomer of each compound was hydrolyzed fourfold faster than the respective (S)-enantiomer. Even though the enzyme inhibition rates of (R)- and (S)-MONO were much slower than those of their respective bambuterol enantiomers (∼15-fold), both MONO enantiomers showed a significant BChE inhibition when physiologically relevant concentrations of enzyme and inhibitors were used (∼50% of their respective bambuterol enantiomers). The kinetic constants obtained by testing each single compound were used to model the contribution given by MONO to the enzyme inhibition observed for bambuterol. The hydrolysis of MONO enantiomers enhanced the inhibitory power of bambuterol enantiomers of about 27.5% (R) and 12.5% (S) and extended more than 1 hour the duration of inhibition. The data indicate that MONO contribute significantly to the inhibition of BChE occurring in humans upon administration of normal doses of bambuterol. In addition, the hydrolysis of MONO resulted in the rate-limiting step in the conversion of bambuterol in its pharmacologically active metabolite terbutaline; therefore, MONO concentrations should always be monitored during pharmacokinetic studies of bambuterol.

Simultaneous determination of bambuterol and its two major metabolites in human plasma by hydrophilic interaction ultra-performance liquid chromatography-tandem mass spectrometry.

In this study, a rapid and sensitive hydrophilic interaction ultra-performance liquid chromatography-tandem mass spectrometry (HILIC-UPLC-MS/MS) method was developed for simultaneous determination of bambuterol and its two major metabolites monocarbamate bambuterol and terbutaline in human plasma. All samples were simply precipitated using acetonitrile and separated on a UPLC-HILIC column under gradient elution with a mobile phase consisting of acetonitrile and water with the addition of 10mm ammonium acetate and 0.1% formic acid at 0.4 mL/min. The analytes were detected by a Xevo TQ-S tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. The established method was highly sensitive with the lower limit of quantification (LLOQ) of 10.00 pg/mL for each analyte, and the intra- and inter-day precisions were <12.8%. The analytical runtime within 4.0 min per sample made this method suitable for high throughput determination. The validated method was successfully applied to a clinical pharmacokinetic study of bambuterol in eight healthy volunteers. Furthermore, the effects of the chromatographic conditions on the retention of the analytes on HILIC were investigated, and the benefits of HILIC were evaluated by comparing with a C18 column. The results indicated that liquid-liquid partition and the electrostatic interactions played an important role in the retention of the analytes on HILIC in this study. And HILIC offered particular advantages over RPLC approach in the aspects of the peak symmetry, the column efficiency, and the column pressure.

An estimation of beta 2-adrenoceptor reserve on human bronchial smooth muscle for some sympathomimetic bronchodilators.

BACKGROUND AND PURPOSE: The beta(2)-adrenoceptor on human pro-inflammatory cells is exquisitely sensitive to desensitization, whereas beta(2)-adrenoceptor-mediated relaxation of human airways smooth muscle (HASM) is relatively resistant to this phenomenon. An explanation for this discrepancy is that a large beta(2)-adrenoceptor 'reserve' exists on HASM cells for sympathomimetic bronchodilators, which protects against desensitization. EXPERIMENTAL APPROACH: The operational model of agonism was used to estimate the affinity of salbutamol, terbutaline, formoterol and procaterol for the beta(2)-adrenoceptors in methacholine (MCh)-contracted HASM from which the relationship between fractional receptor occupancy and relaxation was determined. This analysis was performed under conditions of fractional, irreversible, beta(2)-adrenoceptor inactivation and, for salbutamol and terbutaline only, by the comparative method of Barlow et al. The affinity of salbutamol for the beta(2)-adrenoceptor guinea-pig eosinophils and the receptor/occupancy-response relationship for the suppression of the respiratory burst (an index of pro-inflammatory cell function) was also determined. KEY RESULTS: For salbutamol and terbutaline, both pharmacological approaches yielded in HASM discrepant affinity estimates (values differed, maximally, by 0.67 log(10) unit). However, affinity values more closely agreed (difference <0.47 log(10) unit), when operational analysis was performed on data corrected for 'fade' of the MCh-induced contraction. Plots of fractional beta(2)-adrenoceptor occupancy versus relaxation indicated a receptor 'reserve' for all agonists tested at all levels of response. In contrast, minimal receptor reserve was detected for the ability of salbutamol to suppress respiratory burst activity in eosinophils. CONCLUSIONS AND IMPLICATIONS: These data may help explain the relative inability of sympathomimetic bronchodilators to render HASM tolerant to beta(2)-adrenoceptor-mediated relaxation.

Novel mutants of the human beta1-adrenergic receptor reveal amino acids relevant for receptor activation.

Activation of G protein-coupled receptors like the beta(1)-adrenergic receptor results in conformational changes that ultimately lead to signal propagation through a G protein to an effector like adenylyl cyclase. In this study we identified amino acids that seem to be critical for activation of the human beta(1)-adrenergic receptor. Activation patterns of mutant receptors were analyzed using two structurally different ligands for beta-adrenergic receptors that both are mixed agonist/antagonists. Broxaterol and terbutaline are agonists at beta(2)- and beta(3)-receptors; however, they act as antagonists at the beta(1)-subtype. We reasoned that this functional selectivity may be reflected by a corresponding sequence pattern in the receptor subtypes. Therefore, we exchanged single amino acids of the beta(1)-adrenergic receptor for residues that were identical in the beta(2)- and beta(3)-subtypes but different in the beta(1)-receptor. Pharmacological characterization of such receptor mutants revealed that binding of a panel of agonists and antagonists including broxaterol and terbutaline was unaltered. However, two of the mutants (I185V and D212N) were activated by broxaterol and terbutaline, which acted as antagonists at the wild-type receptor. Two additional mutants (V120L and K253R) could be activated by terbutaline alone, which is structurally more closely related to endogenous catecholamines like epinephrine than to broxaterol. A model of the human beta(1)-adrenergic receptor showed that the four gain-of-function mutations are outside of the putative ligand-binding domain substantiating the lack of an effect of the mutations on binding characteristics. These results support the notion that Val-120, Ile-185, Asp-212, and Lys-253 are critically involved in conformational changes occurring during receptor activation.

Quantitative estimation of myocardial fibrosis based on receptor occupancy for beta2-adrenergic receptor agonists in rats.

To develop beta2-adrenergic receptor (AR) agonists with higher selectivity, it is essential to evaluate the cardiac side effects which are the most serious side effects of this class of drugs. We studied receptor occupancy of beta1-ARs in rats as a possible cause for the side effect of beta2-AR agonists, namely myocardial fibrosis. Myocardial fibrosis in rats was observed on Day 7 after the administration of salbutamol and terbutaline, both of which are selective beta2-AR agonists, at higher dose levels. To evaluate receptor occupancy, plasma concentrations of (R)-salbutamol and (R)-terbutaline, plasma protein binding and the EC50 for chronotropic effects in rats were determined. Based on the plasma concentrations, the plasma protein binding and EC50, receptor occupancy-time profiles were constructed. The relationship between the receptor occupancy-time profile under the curve, the AUCphi, and the degree of myocardial fibrosis was evaluated with a multiple correlation analysis. Myocardial fibrosis was significantly correlated (r2 > 0.78) to the AUCphi with the threshold above approximately 50%, but not to plasma concentrations. These results indicate that the receptor occupancy theory is also useful for the evaluation of the chronotropic side effects of beta2-AR agonists.

Dynamics of beta2-adrenergic receptor-ligand complexes on living cells.

The agonist-induced dynamic regulation of the beta(2)-adrenergic receptor (beta(2)-AR) on living cells was examined by means of fluorescence correlation spectroscopy (FCS) using a fluorescence-labeled arterenol derivative (Alexa-NA) in hippocampal neurons and in alveolar epithelial type II cell line A549. Alexa-NA specifically bound to the beta(2)-AR of neurons with a K(D) value of 1.29 +/- 0.31 nM and of A549 cells with a K(D) of 5.98 +/- 1.62 nM. The receptor density equaled 4.5 +/- 0.9 microm(-2) in neurons (rho(N)) and 19.9 +/- 2.0 microm(-2) in A549 cells (rho(A549)). Kinetic experiments revealed comparable on-rate constants in both cell types (k(on) = 0.49 +/- 0.03 s(-1) nM(-1) in neurons and k(on) = 0.12 +/- 0.02 s(-1) nM(-1) in A549 cells). In addition to the free ligand diffusing with a D(free) of (2.11 +/- 0.04) x 10(-6) cm(2)/s, in both cell types receptor-ligand complexes with two distinct diffusion coefficients, D(bound1) (fast lateral mobility) and D(bound2) (hindered mobility), were observed [D(bound1) = (5.23 +/- 0.64) x 10(-8) cm(2)/s and D(bound2) = (6.05 +/- 0.23) x 10(-10) cm(2)/s for neurons, and D(bound1) = (2.88 +/- 1.72) x 10(-8) cm(2)/s and D(bound2) = (1.01 +/- 0.46) x 10(-9) cm(2)/s for A549 cells]. Fast lateral mobility of the receptor-ligand complex was detected immediately after addition of the ligand, whereas hindered mobility (D(bound2)) was observed after a delay of 5 min in neurons (up to 38% of total binding) and of 15-20 min in A549 cells (up to 40% of total binding). Thus, the receptor-ligand complexes with low mobility were formed during receptor regulation. Consistently, stimulation of receptor internalization using the adenylate cyclase activator forskolin shifted the ratio of receptor-ligand complexes toward D(bound2). Intracellular FCS measurements and immunocytochemical studies confirmed the appearance of endocytosed receptor-ligand complexes in the cytoplasm subjacent to the plasma membrane after stimulation with the agonist terbutaline (1 microM). This regulatory receptor internalization was blocked after preincubation with propranolol and with a cholesterol-complexing saponin alpha-hederin.

Flow-injection chemiluminescence detection for studying protein binding of terbutaline sulfate with on-line microdialysis sampling.

The binding of terbutaline sulfate to bovine serum albumin was studied in vitro using the technique of microdialysis sampling combined with flow-injection chemiluminescence analysis (FIA-CL). In the presence of formaldehyde, terbutaline sulfate can be oxidized by KMnO(4) to produce high chemiluminescence emission in sulfate acid media. The concentration of terbutaline sulfate is proportional with the CL intensity in the range of 1 x 10(-7)-2 x 10(-5) mol l(-1) with a detection limit of 3 x 10(-8) mol l(-1). The drug and protein were mixed in different molar ratios in 0.067 mol l(-1) phosphate buffer, pH 7.4, and incubated at 37 degrees C in a water bath. The microdialysis probe was utilized to sample the mixed solution at a perfusion rate of 5 microl min(-1) and the dialytic efficiency of terbutaline sulfate under the experimental conditions was 26.3%. The data obtained by proposed microdialysis flow-injection chemiluminescence method was analyzed with Scrathard analysis and Klotz plot. The estimated association constant (K) and the number of the binding site (n) on one molecule of BSA by Scrathard analysis were 4.11 x 10(4) l mol(-1) and 1.06, respectively. The proposed system proved that FIA-CL coupled with on-line microdialysis sampling is a simple and reliable technique for the study of drug-protein interaction.

The Ile164 beta(2)-adrenoceptor polymorphism alters salmeterol exosite binding and conventional agonist coupling to G(s).

beta(2)-adrenoceptors (beta(2)AR) are polymorphic at amino acid 164 (Thr or Ile) of the fourth transmembrane domain. In transfected fibroblasts, six agonists commonly used in the treatment of bronchospasm were studied. Isoproterenol, albuterol, metaproterenol, terbutaline, formoterol, and salmeterol displayed decreased binding affinities (K(i)s were 1.2-3.0-fold higher) and a significant degree of impaired maximal stimulation of adenylyl cyclase ( approximately 40%), was observed with all agonists for the Ile164 receptor. The ratios of signal transduction efficiencies (Tau function, Ile164/Thr164) varied from a low of 0.17 for terbutaline to 0.49 for salmeterol. In addition, Ile164 bound salmeterol at the exosite, as delineated in perfusion washout studies, at a decreased level (31+/-4.8% vs. 49+/-4.4% retained salmeterol, respectively, P=0.02). In cAMP production studies under perfusion conditions, this decreased exosite binding caused a approximately 50% decrease in the duration of action of salmeterol at Ile164 (t(1/2)=21.0+/-3.6 vs. 46.8+/-4.1 min for Thr164, P=0.001). The durations of action for isoproterenol and formoterol under similar perfusion conditions were not different between the two receptors. These in vitro results indicate the Ile164 polymorphic receptor represents a pharmacogenetic locus for the most commonly utilized agonists in the treatment of asthma with a unique phenotype for salmeterol.

[Bioequivalence of bambuteral and its metabolites terbutaline after oral bambuteral tablet in healthy volunteers by HPLC/MS].

AIM: To study bioequivalence of bambuteral and its metablites terbutaline in 20 healthy male volunteers. METHODS: A single oral dose of domestic or imported bambuteral tablet was given according to a randomized 2-way cross-over design. The plasma bambuteral and terbutaline concentrations were determined by HPLC/MS. RESULTS: The pharmacokinetic parameters of domestic and imported bambuteral: AUC0-t were (52 +/- 21) microgram.h.L-1 and (51 +/- 20) microgram.h.L-1, Tmax were (2.9 +/- 0.9) h and (2.6 +/- 0.7) h, Cmax were (6.0 +/- 2.6) microgram.L-1 and (6.2 +/- 2.9) microgram.L-1, T1/2Ke were (11.2 +/- 2.3) h and (11.2 +/- 1.9) h, respectively; terbutaline: AUC0-t were (191 +/- 30) microgram.h.L-1 and (197 +/- 37) microgram.h.L-1; Tmax were (4.2 +/- 1.0) h and (4.2 +/- 1.0) h; Cmax were (10 +/- 5) microgram.L-1 and (10 +/- 4) microgram.L-1; T1/2Ke were (20 +/- 3) h and (21 +/- 4) h, respectively. The bioavaiability of the domestics was 102% +/- 8% (bambuteral) and 100% +/- 12% (terbutaline). CONCLUSION: The results demonstrate that the two forms of bambuteral and terbutaline were bioequivalent by analysis of variance, two-one sided test and 90% confidential limit.

Beta2-adrenoceptor agonist suppresses tumour necrosis factor production in rat mesangial cells.

This study aimed to investigate the time-course of the effect of beta2-adrenoceptor stimulation with terbutaline on lipopolysaccharide (LPS)-induced tumour necrosis factor(TNF)-alpha production in rat mesangial cells. Cells were cultured from 0-24 h in the presence of LPS (1 microg/ml) and/or terbutaline (10(-7)-10(-8) mol/l). After 1 h of incubation, terbutaline inhibited TNF-alpha protein release as well as transcription and translation of TNF-alpha and mitogen activated protein kinase (MAPK, p42/p44) activity. At 3 h, terbutaline enhanced intracellular cAMP but suppressed TNF-alpha release and transcription. By 24 h, whereas terbutaline was no longer influencing transcription or translation, TNF-alpha release remained depressed which correlated with an increase in supernatant interleukin (IL)-6. Terbutaline did not affect the LPS-induced IL-10 produced in the cell. These findings indicate that beta2-adrenoceptor stimulation during an LPS challenge prevented TNF-alpha production as a consequence of MAPK inhibition and enhanced cAMP generation, which at a later stage was associated with an anti-inflammatory effect of IL-6.

Pharmacokinetics of bambuterol in subjects homozygous for the atypical gene for plasma cholinesterase.

AIMS: It has been assumed that both plasma cholinesterase (EC 3.1.1.8) and oxidative enzymes are needed for optimum formation of the bronchodilator terbutaline from its biscarbamate prodrug bambuterol. The present study aimed at investigating the fate of bambuterol in subjects with deficient plasma cholinesterase but with normal oxidative (CYP2D6) capability. METHODS: The pharmacokinetics of bambuterol and terbutaline were studied in four healthy subjects (two men and two women) being homozygous for the atypical gene for plasma cholinesterase. Their oxidative metabolism was apparently good as they were all rapid metabolizers of debrisoquine. Bambuterol hydrochloride 20 mg was given orally once daily for 10 days, and plasma and urine samples were taken for 1.5 days (plasma) and 4.5 days (urine) after administration of the last dose. RESULTS: The pharmacokinetic parameters in the present study were grossly similar to those found in a study of bambuterol in subjects with normal plasma cholinesterase activity (N). However, subjects with atypical cholinesterase had a shorter terminal half-life of bambuterol (a measure of uptake rate), 4.8-12.6 h vs 8.3-22.3 h in N, and slightly higher plasma concentrations of bambuterol (average concentrations 1.9-3.7 nmol l(-1) vs 1.5-3.1 nmol l(-1) in N). Peak/trough terbutaline plasma concentrations ratios (2.1-3.2) were somewhat increased, but average plasma concentrations (8.3-14.5 nmol l(-1)) and terminal half-life (16.5-21.8 h) of terbutaline did not differ. CONCLUSIONS: In Caucasian populations, one subject out of 2500 is homozygous for the atypical gene for plasma cholinesterase. The atypical enzyme has a much lower affinity for bambuterol than the normal enzyme. Nevertheless, the subjects with atypical cholinesterase were able to produce terbutaline as efficiently as normal subjects. This might be explained by an altered uptake and metabolism in the absence of plasma cholinesterase, or the importance of this enzyme for the formation of terbutaline from bambuterol in vivo may have been overestimated.