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The locus coeruleus (LC) provides the primary noradrenergic input to the forebrain and hippocampus, and may be vulnerable to degeneration and contribute to age-related cognitive decline and neuroinflammation. Additionally, inhibition of noradrenergic transmission by brain-permeable beta-blockers could exacerbate cognitive impairment. This study examined effects of age and acute beta-blocker administration on LC and hippocampus pathology, neuroinflammation and learning and memory behavior in mice. Male mice, 3 and 18 months old, were administered propranolol (beta-blocker) or mabuterol (beta-adrenergic agonist) acutely around behavioral assessment. Terminal inflammatory markers in plasma, hippocampus and LC were assessed alongside histopathology. An increase in hippocampal and LC microgliosis and inflammatory proteins in the hippocampus was detected in aged mice. We report pathological hyperphosphorylation of the postsynaptic NMDA receptor subunit 2B (NR2B) in the hippocampus, suggesting neuronal hyperexcitability. Furthermore, the aged proteome revealed an induction in proteins related to energy metabolism, and mitochondria dysfunction in the LC and hippocampus. In a series of hippocampal dependent behavioral assessment tasks acute beta-adrenergic agonist or beta blocker administration altered learning and memory behavior in both aged and young mice. In Y-maze, propranolol and mabuterol differentially altered time spent in novel versus familiar arms in young and aged mice. Propranolol impaired Novel Object Recognition in both young and aged mice. Mabuterol enhanced trace learning in fear conditioning. Aged mice froze more to context and less to cue. Propranolol impaired contextual recall in aged mice. Concluding, aged mice show LC and hippocampus pathology and heightened effects of beta-adrenergic pharmacology on learning and memory., (Copyright © 2021. Published by Elsevier Inc.)

In this work, a novel sulfated-β-cyclodextrin (S-β-CD) coated stationary phase was prepared for open-tubular capillary electrochromatography (OT-CEC). The capillary was developed by attaching polydopamine/sulfated-β-cyclodextrin (PDA/S-β-CD) onto the gold nanoparticles (AuNPs) coated capillary which was pretreated with polydopamine. The results of scanning electron microscopy (SEM) and energy dispersive X-ray analysis spectroscopy (EDS) indicated that polydopamine/sulfated-β-cyclodextrin was successfully fixed on the gold nanoparticles coated capillary. To evaluate the performance of the prepared open tubular (OT) column, the enantioseparation was carried out by using ten chiral drugs as model analytes. Under the optimal conditions, salbutamol, terbutaline, trantinterol, tulobuterol, clorprenaline, pheniramine, chlorpheniramine, brompheniramine, isoprenaline and tolterodine were baseline separated with the resolution (Rs) values of 3.25, 1.76, 2.51, 1.89, 3.17, 2.17, 1.99, 1.72, 2.01 and 3.20, respectively. Repeatability of the column was studied, with the relative standard deviations for run-to-run, day-to-day and column-to-column lower than 5.7%., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This study is aimed to investigate whether Mabuterol (Mab) inhibits proliferation of airway smooth muscle cells (ASMCs) induced by platelet-derived growth factor BB (PDGF-BB) and how far it is related to mitochondrial fission/fusion and intracellular calcium if it comes into play. To explore the mechanism of Mab's antagonizing the proliferation, Mdivi-1, DRP1 inhibitor, which has an inhibitory effect on mitochondrial fission, is used to compare with Mab. Cell viability was measured by either MTT or CCK-8. The inhibitory effect of Mab on S phase of ASM cell cycle induced by PDGF-BB was analyzed by flow cytometry (FCM). Fluo-3/AM, Ca 2+ fluorescent probe, was used to detect Ca 2+ fluorescence intensity by inverted microscope and flow cytometry. The gene expression of Drp-1 and Mfn-2 was observed with Real time PCR and the proteins of Drp-1, Mfn-2, PCNA and cyclin D1 were assessed by Western Blot. Mab and Mdivi-1 both suppressed the proliferation induced by PDGF-BB. The results from inverted microscope and flow cytometry showed that Mab inhibited [Ca 2+ ]i in rat ASMCs induced by PDGF-BB. Cell cycle concept map illustrated that Mab significantly controlled the S phase of ASM cell cycle induced by PDGF-BB. As a consequence, Real time PCR and Western blot revealed the fact that Mab decreased the expression of Drp-1 mRNA and protein, and promoted the expression of Mfn-2 mRNA and protein. These findings suggested that Mab placed restrictions on the proliferation of rat ASMCs induced by PDGF-BB and the mechanism might be associated with the intracellular calcium inhibited and the mitochondrial fission/fusion regulated by Mab., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A selective analytical method based on packed-fiber solid-phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry (PFSPE-UPLC-MS/MS) has been developed for determination of six β-agonists (clorprenaline, bambuterol, clenbuterol, brombuterol, mabuterol, and penbuterol) in pork tissue. Polystyrene-polymeric crown ether (PS-PCE) composite nanofibers were fabricated by electrospinning and utilized to prepare the homemade extraction columns. With optimal conditions, all analytes were separated very well and the blank pork did not disturb the determination, and the linearity is good in a range of 5.0μg/kg-25.0μg/kg. The recoveries were 79.3-110.1%. RSDs for intra-day were in the range of 1.5-10.5% and RSDs for inter-day were 4.7-11.8%. Above all, only 5mg of sorbent and 200μL of elution solvent were favorable to directly extract all analytes in a complex matrix. The method is simple and cost-effective, and has the potential to be applied to quantitatively analyze the concentrations of polar species in food samples containing complex matrix., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Three stable and simple synthetic routes of labeled D 9 -Mabuterol, D 9 -Bambuterol, and D 9 -Cimbuterol were described with 98.5%, 99.7%, and 98.4% isotopic abundance and good purity. These structures and isotope-abundance were confirmed according to 1 H NMR and liquid chromatography-tandem mass spectrometry., (Copyright © 2016 John Wiley & Sons, Ltd.)

This report presents an efficient synthesis of D 6 -clenproperol and D 6 -cimaterol with 99.5% and 99.7% isotopic abundance in acceptable yields and excellent chemical purities with deuterium isopropylamine as labelled precursor. Their structures and the isotope-abundance were confirmed by proton nuclear magnetic resonance and liquid chromatography-mass spectrometry., (Copyright © 2016 John Wiley & Sons, Ltd.)

A highly rapid, selective and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to simultaneously determine trantinterol, its major phase-I metabolites and their primary conjugated metabolites in human plasma. Waters Oasis HLB C18 solid phase extraction cartridges were used in the sample preparation. The separation was carried out on an ACQUITY UPLC™ BEH C18 column with methanol/0.2% formic acid (30:70, v/v) as the mobile phase at a flow rate of 0.25 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in selective reaction monitoring (SRM) mode with the use of an electrospray ionization (ESI) source. The linear calibration curves for trantinterol, tert-butyl hydroxylated trantinterol (tert-OH-trantinterol) and 1-carbonyl trantinterol (trantinterol-COOH) were obtained in the concentration ranges of 0.200-250, 0.108-4.00 and 0.0840-5.02 ng/mL, respectively (r(2)≥0.99). The intra- and inter-day precision (relative standard deviation, RSD) values were less than 13%, and the accuracy (relative error, RE) was within ±9.9%, as determined from quality control (QC) samples for the analytes. The concentrations of conjugated forms of trantinterol and tert-OH- trantinterol in plasma were determined using selective enzyme hydrolysis. The method described herein was fully validated and successfully applied for the pharmacokinetic study of trantinterol in healthy volunteers after oral administration., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objective: The aim of this study was to investigate and compare the pharmacokinetics of trantinterol and its active amphoteric carboxylic acid metabolite (1-carbonyl trantinterol) between the healthy elderly and young subjects., Methods: This was a single-center, open-label, parallel-group study completed by 22 healthy subjects (≥65 years (the elderly group); 18-45 years (the young group); 9 males and 2 females per age group) receiving single oral dose of 50 μg trantinterol tablets. Blood samples were taken at intervals up to 48 hours post-dose., Results: In both groups, maximum plasma concentration of trantinterol was researched at 0.9 hours, while the tmax of 1-carbonyl trantinterol differed slightly. Trantinterol Cmax and AUClast were higher in the elderly group than the young group, by 27% (90% CI, 0.95-1.69) and 77% (90% CI, 1.25-2.51), respectively. For 1-carbonyl trantinterol, Cmax, and AUClast were also higher, by 36% (90% CI, 1.04-1.78) and 71% (90% CI, 1.27-2.30), respectively, in the elderly group. The CL/F and V/F of trantinterol and 1-carbonyl trantinterol were significantly lower in the elderly group, while t1/2 of both did not show significant differences. CL/F of trantinterol and 1-carbonyl trantinterol were found to significantly correlate inversely with age, and positively with the baseline creatinine clearance., Conclusions: A single dose of 50 μg trantinterol was well tolerated. Significant changes in Cmax and AUC of trantinterol and 1-carbony trantinterol were seen in the elderly and may be clinically important.

A highly selective and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of trantinterol and one of its major metabolites, 1-carbonyl trantinterol, in human plasma. An Oasis MCX 96-well solid-phase extraction cartridge and a SeQuantTM ZIC(®)-HILIC LC column were used for sample preparation and chromatographic separation, respectively. The analytes were monitored by a QTrap 5500 mass spectrometer with positive electrospray ionization. Multiple reaction monitoring was used for quantification using the precursor to product ion pairs of m/z 311.1 → 237.9 (trantinterol), m/z 325.1 → 251.9 (1-carbonyl trantinterol) and m/z 368.4 → 294.0 (bambuterol as internal standard). The assay had a calibration range from 0.2 to 50 pg/mL and a lower limit of quantification of 0.2 pg/mL for both trantinterol and 1-carbonyl trantinterol. The inter-day and intra-day precisions were <12.0% and the accuracies were within the range of 87.1-111%. The mean recovery ranged from 82.0 to 97.7% and internal standard normalized matrix effect from 0.813 to 0.899. The analytes were stable under all tested conditions. This validated method was successfully applied to a pilot pharmacokinetic study in healthy subjects administered a single 50 μg oral dose., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Trantinterol is a novel β2-adrenoceptor agonist, currently undergoing clinical trials for the treatment of asthma. We developed and validated an liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of trantinterol and its major metabolite, 1-carbonyl trantinterol (SPFFCOOH), in rat plasma. Aliquots (100μL) of heparinized plasma samples were processed by protein precipitation with acetonitrile. Chromatographic separation used an Acquity UPLC BEH C18 column (2.1mm×50mm, 1.7μm) and acetonitrile-0.1% formic acid (20:80, v/v) as mobile phase, at a flow rate of 0.25mL/min. The detection was performed on a triple-quadrupole tandem mass spectrometer with multiple-reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The precursor-to-product ion transitions m/z 310.9→m/z 237.9 for trantinterol, m/z 324.9→m/z 251.9 for SPFFCOOH and m/z 368.0→m/z 294.0 for bambuterol (internal standard, IS) were used for quantification. The calibration curves were obtained in the concentration of 0.25-100ng/mL for both trantinterol and SPFFCOOH. The intra- and inter-day precision (relative standard deviations, RSD) values were below 15% and accuracy (relative error, RE) was from -4.3% to 6.6% at all quality control (QC) levels. The method was successfully applied to compare the pharmacokinetics of trantinterol and SPFFCOOH in male and female Wistar rats after a single oral administration of trantinterol., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A rapid, selective and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to simultaneously determine trantinterol and its major metabolites in human urine. Waters Oasis HLB C18 solid phase extraction cartridges were used in the urine sample preparation. The separation was carried out on an ACQUITY UPLC™ BEH C18 column with methanol-0.2% formic acid (30:70, v/v) as the mobile phase at a flow rate of 0.25mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The linear calibration curves for trantinterol, arylhydroxylamine trantinterol (N-OH-trantinterol), the tert-butyl hydroxylated trantinterol (tert-OH-trantinterol) and the 1-carbonyl trantinterol (trantinterol-COOH) were obtained in the concentration range of 0.414-207, 0.578-385, 0.168-84.0, and 0.954-477ng/mL, respectively. The linear correlation coefficients were greater than 0.990. The intra and inter-day precision (relative standard deviation, RSD) values were less than 12% and the accuracy (relative error, RE) was 6.7-11%. The method herein described was superior to previous methods in sample throughput and sensitivity and successfully applied to the human excretion study., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A sensitive and enantioselective vancomycin chiral stationary phase high-performance liquid chromatography-tandem mass spectrometry method was developed for the determination of trantinterol enantiomers in human plasma. Baseline resolution was achieved using the vancomycin chiral stationary phase known as Chirobiotic V with polar ionic mobile phase consisting of acetonitrile-methanol (60:40, v/v) containing 0.01% ammonia and 0.02% acetic acid at a flow rate of 1.0 mL/min. Waters Oasis HLB C18 solid phase extraction cartridges were used in the sample preparation of trantinterol samples from plasma. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring mode via electrospray ionization. The calibration curve was linear in a concentration range from 0.0606 to 30.3 ng/mL in plasma, with the lower limit of quantification of 0.0606 ng/mL. The intra- and interday precision (relative standard deviation) values were within 9.7% and the accuracy (relative error) was from -6.6 to 7.2% at all quality control levels. The method was successfully applied to a study of stereoselective pharmacokinetics in human., (© 2015 Wiley Periodicals, Inc.)

P-glycoprotein (P-gp)-mediated drug-drug interactions are important factors causing adverse effects of drugs in clinical use. The aim of this study was to determine whether trantinterol (also known as SPFF), a novel β2-adrenoceptor agonist, was a P-gp inhibitor or substrate. The results showed that trantinterol was not a substrate of P-gp but increased rhodamine 123 (Rho 123) uptake by MDCK-MDR1 cells and decreased the efflux transport of both Rho 123 and cyclosporine A (CsA) in bidirectional transport studies across MDCK-MDR1 cell monolayers. This suggested that trantinterol was a P-gp inhibitor but not a P-gp substrate. The mechanism of inhibition was investigated in the P-gp-Glo assay system, where it was found that trantinterol inhibited P-gp ATPase activity in a dose-dependent manner. A subsequent study using the antibody binding assay with the conformation-sensitive P-gp-specific antibody UIC2 confirmed that trantinterol decreased UIC2 binding at 10 μM in contrast to the competitive inhibitor, verapamil. This suggested that trantinterol was a noncompetitive inhibitor of P-gp. Finally, a pharmacokinetic study in rat showed that trantinterol significantly increased the area under the plasma concentration-time curve (AUC) and maximum plasma concentration (Cmax) of digoxin and paclitaxel (PAC), and the Cmax of cyclosporine A (CsA). In summary, trantinterol is a potent noncompetitive P-gp inhibitor which may increase the bioavailability of other P-gp substrate drugs coadministered with it.

The chiral inversion and pharmacokinetics of two enantiomers of trantinterol, a new β2 agonist, were studied in rats dosed (+)- or (-)-trantinterol separately. Plasma concentrations of (+)- and (-)-trantinterol were measured by chiral stationary phase liquid chromatography tandem mass spectroscopy (LC-MS/MS). The apparent inversion ratio was calculated as the ratio of AUC0-t of (-)-trantinterol or (+)-trantinterol inverted from their antipodes to the sum of the AUC0-t of (-)- and (+)-trantinterol. Following single intravenous administration, both given enantiomers declined in similar plasma concentrations, suggesting that the two enantiomers have approximately the same disposition kinetics by the route of intravenous administration. However, after single oral administration, plasma concentrations of uninverted (-)-trantinterol at many timepoints were significantly higher than those of uninverted (+)-trantinterol, suggesting that the two enantiomers undergo apparently different absorption or metabolism after oral administration. Significant bidirectional chiral inversion occurred after intravenous and oral administration of (+)- or (-)-trantinterol. After dosing with optically pure enantiomer, the concentration of the administered enantiomer predominated in vivo. The AUC0-36 of (+)-trantinterol after intravenous and oral dosing of (-)-trantinterol were 16.6 ± 5.2 and 33.3 ± 16%, respectively of those of total [(+) + (-)] trantinterol. The AUC0-36 of (-)-trantinterol after intravenous and oral dosing of (+)-trantinterol were 19.6 ± 8.8 and 37.9 ± 4.5%, respectively, of those of total [(-) + (+)] trantinterol. After intravenous administration of (+)- and (-)-trantinterol the chiral inversion ratios of the two enantiomers were not significantly different and similar results were found for oral administration. The extent of chiral inversion after intravenous administration was apparently lower, indicating that the bidirectional chiral inversion was not only systemic but also presystemic., (© 2013 Wiley Periodicals, Inc.)

A highly selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of trantinterol (SPFF) and its major metabolites for the first time. The analytes were extracted from rat urine and feces samples by liquid-liquid extraction (LLE) and determined in multiple reaction monitoring (MRM) mode with clenbuterol as the internal standard. Chromatographic separation was achieved on a Venusil ASB C8 column (2.1mm×100mm, 3μm), with the mobile phase consisted of methanol-0.2% formic acid (30:70, v/v) at the flow rate of 0.2mL/min. Each sample was chromatographed within 5min. This method has a lower limit of quantification (LLOQ) of 0.450, 1.05, 1.35, 0.904 and 1.36ng/mL for trantinterol (SPFF), arylhydroxylamine trantinterol (N-OH-SPFF), tert-butyl hydroxylated trantinterol (Tert-OH-SPFF), 1-carbonyl trantinterol (SPFF-COOH) and 3-methyl sulfone-dechloro-trantinterol (SPFF-SO2CH3) in rat urine, and 0.450, 1.35 and 0.904ng/mL for SPFF, Tert-OH-SPFF and SPFF-COOH in rat feces, respectively. The linear correlation coefficients were greater than 0.990. The intra- and inter-day precision (relative standard deviation, RSD) values were below 15% and the accuracy (relative error, RE) was -9.9% to 11% at three quality control levels. The method has been successfully applied to the excretion study following an oral administration of 1mg/kg trantinterol to rats., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Trantinterol is a novel β(2)-adrenoceptor agonist used for the treatment of asthma. The aim of this study is to identify the metabolites of trantinterol using liquid chromatography tandem mass spectrometry (LC-MS/MS), to isolate the main metabolites, and confirm their structures by nuclear magnetic resonance (NMR). Urine, feces, bile, and blood samples of rats were obtained and analyzed. Reference standards of six metabolites were achieved with the combination of chemical synthesis, microbial transformation, and the model systems of rats. Moreover, in order to investigate the phase I metabolism of trantinterol in humans and to study the species differences between rats and humans, incubations with liver microsomes were performed. The biotransformation by a microbial model Cunninghamella blakesleana AS 3.970 was also studied. A total of 18 metabolites were identified in vivo and in vitro together, 13 of which were newly detected. Three phase I metabolites were detected in vivo and in vitro as well as in the microbial model, including the arylhydroxylamine (M1), the tert-butyl hydroxylated trantinterol (M2) and the 1-carbonyltrantinterol (M3). Another important pathway in rats is glutathione conjugation and further catabolism and oxidation to form consecutive derivatives (M4 through M10). Other metabolites include glucuronide, glucoside, and sulfate conjugates. The results of in vitro experiments indicate no species difference exists among rats, humans, and C. blakesleana AS 3.970 on the phase I metabolism of trantinterol. Our study provided the most comprehensive picture for trantinterol in vivo and in vitro metabolism to this day, and may predict its metabolism in humans.

Dispersive liquid-liquid microextraction based on solidification of floating organic drop (DLLME-SFO) was for the first time combined with field-amplified sample injection (FASI) in CE to determine four β(2)-agonists (cimbuterol, clenbuterol, mabuterol, and mapenterol) in bovine urine. Optimum BGE consisted of 20 mM borate buffer and 0.1 mM SDS. Using salting-out extraction, β(2)-agonists were extracted into ACN that was then used as the disperser solvent in DLLME-SFO. Optimum DLLME-SFO conditions were: 1.0 mL ACN, 50 μL 1-undecanol (extraction solvent), total extraction time 1.5 min, no salt addition. Back extraction into an aqueous solution (pH 2.0) facilitated direct injection of β(2)-agonists into CE. Compared to conventional CZE, DLLME-SFO-FASI-CE achieved sensitivity enhancement factors of 41-1046 resulting in LODs in the range of 1.80-37.0 μg L(-1). Linear dynamic ranges of 0.15-10.0 mg L(-1) for cimbuterol and 15-1000 μg L(-1) for the other analytes were obtained with coefficients of determination (R(2)) ≥ 0.9901 and RSD% ≤5.5 (n = 5). Finally, the applicability of the proposed method was successfully confirmed by determination of the four β(2)-agonists in spiked bovine urine samples and accuracy higher than 96.0% was obtained., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A simple, sensitive, and rapid method for determination of L-trantinterol in rat plasma was developed for the first time by using LC coupled to MS/MS based on chiral stationary phase. A baseline separation of the enantiomers of trantinterol was achieved on a Chirobiotic V column, using a mixture of acetonitrile-methanol-ammonia-acetic acid (80:20:0.01:0.02, v/v/v/v) as the mobile phase. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring mode via ESI. The calibration curve was linear in concentration range from 0.270 to 108 ng/mL in plasma with the lower limit of quantification of 0.270 ng/mL. The intra- and interday precision (relative standard deviation) values were within 10.9% and the accuracy (relative error) was from 2.6 to 9.2% at all quality control levels. The method has been successfully applied to a study of L-trantinterol pharmacokinetics in rats., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A high performance liquid chromatographic (HPLC) method using cellulose tris-(3,5-dimethylphenylcarbamate) as chiral stationary phase (Lux Cellulose-1) for the separation of trantinterol hydrochloride enantiomers was developed. The method was based on normal phase model with n-heptane as the basal solvent of mobile phase. The influences of organic modifiers, mobile phase additives and column temperature on the retention and separation of the enantiomers were examined and discussed. It was demonstrated that the mobile phase additives had a dominant effect on the enantiomeric separation. No separation was observed with the addition of diethylamine only in the mobile phase, while the retention was increased and the enatiomeric separation was observed with the addition of trifluoroacetic acid. When both trifluoroacetic acid and diethylamine were added into the mobile phase, the enantioseparation was significantly improved with a resolution up to 4.0. Ethanol and isopropanol were investigated as the organic modifiers, and ethanol offered a better enatiomeric resolution for trantinterol hydrochloride. In the examined temperature range between 15 degrees C and 35 degrees C both separation factor and resolution were decreased with the increase of the column temperature. The optimized chiral HPLC method for the separation of trantinterol hydrochloride enantiomers involved a Lux Cellulose-1 column (250 mm x 4.6 mm, 5 microm), a mobile phase of n-heptane/ethanol/trifluoroacetic acid/diethylamine (88: 12: 0.3: 0.05, v/v/v/v) at a flow rate of 1.0 mL/min, a detection at 246 nm and a column temperature of 25 degrees C. The method is simple and rapid for the enantiomeric impurity determination of (-)-trantinterol hydrochloride bulk samples.

This paper describes a method for the determination of priority β-agonists in urine based on a fully automated sample preparation procedure using an online TurboFlow™ chromatography clean-up step and determination with Orbitrap™ mass analyser technology. The principle of the method was the enrichment of the β-agonists after enzymatic hydrolysis overnight on a small column packed with a special stationary phase (TurboFlow™) while flushing away sample matrix and interfering compounds. Thereafter, the analytes were transferred onto an analytical column and detected by liquid chromatography/high-resolution mass spectrometry in full-scan mode at a resolution of R = 50,000 FWHM (full width at half maximum) and in higher energy collisional dissociation (HCD) scan mode at a resolving power of 10,000 FWHM. The optimisation of each step of the method, such as selection of the TurboFlow™ and analytical column as well as sample loading and elution parameters were performed using a standard solution containing salbutamol, clenbuterol and mabuterol at a concentration of 100 µg l(-1). The developed automated sample preparation significantly improved the throughput and efficiency of the previously used screening method and it resulted in a considerable reduction in analysis time. Validation experiments including 24 β-agonists in urine gave decision limits (CCα) between 0.05 and 0.35 µg l(-1). The repeatability of analyses for urine samples spiked at 0.5 µg l(-1) was within the range of 5-26% and recoveries for all compounds were within 89-107%.

The effect of a novel β(2)-adrenoceptor agonist, trantinterol on the activities of cytochrome P450 (CYP450) was investigated with human liver microsomes and human cryohepatocytes in order to assess the potential for drug-drug interactions. The ability of trantinterol to inhibit CYP450 activities was evaluated in vitro in human liver microsomes. Trantinterol did not inhibit CYP2C19, CYP2D6, and CYP3A4/5 (IC(50)>100 μM). It acted as a weak inhibitor of CYP1A2 and CYP2C9 with IC(50) of 70.8 and 81.9 μM, respectively. No time-dependent inhibitions were observed in the present research. To evaluate CYP450 induction, human cryohepatocytes (n=3) were used and treated once daily for 3 days with trantinterol (0.01, 0.1, and 1 ng/ml), after which CYP450 activities were measured. At concentration of 0.01 ng/ml, which is close to the C(max) at maximal recommended doses (50 μg), trantinterol was about 8% as effective as omeprazole (CYP1A2 inducer) only with donor 2. At concentration of 1 ng/ml, trantinterol was about 3.6 ± 3.1% as effective as rifampin (CYP3A4/5 inducer). These in vitro results indicated that, at pharmacological relevant concentrations, trantinterol will not produce clinically significant CYP450 inhibition or induction., (Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.)

Objective: To examine whether beta2-adrenergic agonist-induced hypertrophy of the quadriceps skeletal muscle can modulate the severity of osteoarthritis (OA) in the rodent meniscectomy (MNX) model., Methods: Male Lewis rats were subcutaneously administered with 1.5 mg/kg/day clenbuterol hydrochloride (n=15) or saline vehicle (n=20) for 14 days. Following pre-treatment, five animals from each group were sacrificed to assess the immediate effects of clenbuterol. The remaining animals underwent either invasive knee surgery (clenbuterol pre-treated n=10; saline pre-treated n=10) or a sham control surgical procedure (saline pre-treated n=5). During disease initiation and progression, weight bearing was assessed by hindlimb loading. Myosin heavy chain (MHC) protein isoforms were quantified by silver stained SDS PAGE. OA severity was graded by assessment of toluidine blue stained step coronal sections of the total knee joint., Results: Clenbuterol treatment resulted in an increase in total bodyweight, growth rate and in quadriceps skeletal muscle mass. Meniscal surgery resulted in the development of OA-like lesions, changes to weight bearing, and changes in MHC protein expression in the quadriceps. Clenbuterol-induced skeletal muscle hypertrophy had no effect on either weight bearing or articular pathology following MNX surgery., Conclusions: Our data reveal that clenbuterol-induced skeletal muscle hypertrophy is unable to mimic the beneficial clinical effects of increased musculature derived through targeted strength training in humans, in a rodent model of MNX-induced OA. In addition we observed fibre-type switching to "slow twitch" in the quadriceps muscle during the induction of OA that warrants further investigation as to its relationship to joint stability., (2010 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

A liquid chromatography-tandem mass spectrometry method was developed for the identification of metabolites of trantinterol, a novel beta(2)-adrenoceptor agonist, in beagle dog urine. The separation of metabolites was performed on a reversed-phase C(8) column using 0.1% formic acid in water and methanol (70 : 30, v/v) as the mobile phase. The structural information and elemental information of metabolites were acquired by an electrospray ionization tandem mass spectrometer and a quadrupole time-of-flight mass spectrometer, respectively. A total of 13 metabolites were detected and characterized on the basis of their tandem MS/MS fragmentation patterns. The accurate masses of nine metabolites were determined and two metabolites were further confirmed by comparing with reference standards. The metabolic pathways of trantinterol in beagle dog are proposed., (2009 John Wiley & Sons, Ltd.)

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of trantinterol enantiomers in rat plasma. Diphenhydramine was employed as the internal standard. The plasma samples were prepared using liquid-liquid extraction with n-hexane-dichloromethane-isopropanol (20:10:1, v/v/v) as the extractant. Trantinterol enantiomers after pre-column derivatization using diacetyl-l-tartaric anhydride (DATAAN) were separated on a C18 column using a gradient solvent programme. The mobile phase was composed of 3mM ammonium acetate and acetonitrile. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI). Linear calibration curve for each enantiomer was obtained in the concentration range of 1-80 ng/mL, with limit of quantification (LOQ) of 1 ng/mL. The intra- and inter- precision (R.S.D.) values were below 9.6% and accuracy (R.E.) was from -2.4 to 6.2% at all quality control (QC) levels. The developed method was applied to the enantioselective pharmacokinetic study of trantinterol in rats.

A rapid and sensitive assay for trantinterol, a novel beta(2)-adrenoceptor agonist, in human plasma has been developed. Samples containing the analyte and internal standard, clenbuterol, were analyzed by liquid chromatography-tandem mass spectrometry after liquid-liquid extraction with diethyl ether:dichloromethane (60:40, v/v). Separation was performed on a Venusil MP C(18) column (50 mm x 4.6 mm, 5 microm) using methanol:1% formic acid (50:50, v/v) as mobile phase and monitored by multiple reaction monitoring of the precursor-to-product ion transitions of trantinterol at m/z 311.2-->238.1 and clenbuterol at m/z 277.2 --> 203.1. The total run time was only 1.5 min and the method was linear over the concentration range 1-1000 pg/mL with a lower limit of quantitation of 1 pg/mL. Intra- and inter-day precisions (relative standard deviation) were below 7% and 12%, respectively, with accuracy (relative error) below 8%. The method was successfully applied to a pharmacokinetic study involving oral administration of a 50 microg trantinterol tablet to healthy volunteers.

The control of illegal use of clenbuterol and other beta(2)-agonist drugs as growth promoters in the European Union countries has led to outlaw practices for synthesizing new concept molecules, showing similar biological activity but not detectable by test methods usually employed to perform the official monitoring programmes. The synthesis schemes of some beta(2)-agonist compounds, formally derived from clenbuterol, were found out by Italian detective authorities. These compounds were synthesised ex novo in our laboratories: then, both their molecular structures and biological activities were characterised. In this paper, we describe different strategies for purifying some beta(2)-agonist drugs of new concept, more hydrophobic than clenbuterol. A two-step clean up procedure, prior to gas chromatography-mass spectrometry analysis, was developed for the multi-residue determination of these beta(2)-agonists from bovine hair and urine. The purification strategy we chose was based on adsorption solid phase extraction and, subsequently, on specific molecular recognition by affinity chromatography. The affinity columns were homemade by coupling bovine alpha(1)-acid glycoprotein, a plasmatic acceptor for basic drugs, to a chromatographic support; their effectiveness for purifying new beta(2)-agonists was discussed. The data about method recoveries and repeatability were also reported.

The enantioselective pharmacokinetics of mabuterol was studied in six rats after single oral dose administration of mabuterol racemate. Serial plasma samples were collected and the pharmacokinetic behavior of each enantiomer in rats was characterized using a sequential achiral and chiral liquid chromatographic method. This method involved the separation of mabuterol racemate from endogenous substances on an achiral ODS column and enantiomeric separation on a Chirobiotic V column. The plasma-concentration data were analyzed for individual mabuterol enantiomer using 3P97 software. After i.g. administration of mabuterol racemate at a dose of 10 mg/kg, both enantiomers were slowly absorbed, reaching mean C(max) of 266.8 and 277.9 ng/mL at t(max) of 5.3 and 5.7 h for R- and S-mabuterol, respectively. The AUC(0-infinity) (5,938.9 ng h/mL) of R-mabuterol was significantly higher than that (4,446.1 ng h/mL) of S-mabuterol, and the half-life (14.5 h) was longer than that (9.6 h) of S-mabuterol (p < 0.001 and p < 0.01, respectively), showing that enantioselective pharmacokinetics between mabuterol enantiomers occur during the metabolism phase.

A multiresidue method was developed and validated to screen bovine urine samples for 10 beta-2-adrenergic agonistic drugs--brombuterol, cimaterol, clenbuterol, clenpenterol, isoxsuprine, mabuterol, ractopamine, ritodrine, salbutamol, and tulobuterol--at the 2 microg/L level. The method is also quantitative in the range of 1 to 4 microg/L for all analytes except salbutamol. The procedure uses enzymatic digestion, liquid-liquid extraction, and cleanup on solid-phase extraction columns, followed by detection using a liquid chromatograph-tandem quadrupole mass spectrometer operated in the positive-ion atmospheric pressure chemical ionization multiple-reaction monitoring mode. Method validation included assessment of recoveries, repeatabilities, linearity of responses, decision limits, and detection capabilities. Overall average recoveries ranged from 70-91%; recoveries were generally lower for salbutamol. The decision limits ranged from 0.4-1.0 microg/L, and detection capabilities from 0.6-1.7 microg/L.

Several novel thiol-reactive clenbuterol analogues were coupled in high yield with bovine serum albumin (BSA). After labelling of unreacted cysteines with maleimide spin label (MiSL), the yield of the coupling reaction was determined by electron paramagnetic resonance (EPR) spectroscopy and spectral analysis. Two spin-probe populations with different mobility states were quantitatively determined. Molecular dynamics was used to model the structure of clenbuterol analogues and spin label conjugated to BSA and recognition of conjugates by anti-clenbuterol antibodies was demonstrated. The recognition of BSA-A, BSA-C and BSA-S conjugates with monoclonal and polyclonal anti-clenbuterol (mCLB-Ab and rCLB-Ab) antibodies was an indication, that chlorine substituents on the aromatic ring of clenbuterol derivatives are not necessary for the binding of antibodies to the conjugates. These results confirmed the importance of the tert-butylamino group as a part of the epitope and contribute to the understanding of the recognition process with anti-clenbuterol antibodies.

Electrospray ionization (ESI) mass spectra of 19 common beta(2)-agonists were investigated in terms of fragmentation pattern and dissociation behavior of the analytes, proving the origin of fragment ions and indicating mechanisms of charge-driven and charge-remote fragmentation. Based on these data, liquid chromatographic/ESI tandem mass spectrometric (LC/ESI-MS/MS) screening and confirmation methods were developed for doping control purposes. These procedures employ established sample preparation steps including either acidic or enzymatic hydrolysis, alkaline extraction and, in the case of equine urine specimens, acidic re-extraction of the analytes. In addition, a degradation product of formoterol caused by acidic hydrolysis during sample preparation could be identified and utilized as target compound in screening and also confirmation methods. The screening procedures cover 18 or 19beta(2)-agonists, the estimated limits of detection of which for equine and human urine samples vary between 2 and 100 ng ml(-1) and between 2 and 50 ng ml(-1), respectively. A single LC/MS/MS analysis can be performed in 9 min., (Copyright 2003 John Wiley & Sons, Ltd.)

The only free thiol group of bovine serum albumin (BSA) was coupled in a high yield with some novel thiol-reactive clenbuterol analogues. The unreacted cysteines were probed with maleimide spin label to determine the yield of the coupling reaction. A novel approach to determining free thiol groups of BSA quantitatively by electron paramagnetic resonance spectroscopy and spectral decomposition without the usual gel-filtration step or extensive dialysis is presented., (Copyright 2003 Elsevier Science (USA))

Previously, we demonstrated the involvement of Asn293 in helix VI of the human beta(2)-adrenergic receptor in stereoselective agonist recognition and activation. In the present study, we have further explored the role of this residue by synthesizing derivatives of isoproterenol and clenbuterol, two beta-adrenergic receptor agonists. We analyzed their efficacy and affinity on the wild-type and a mutant receptor (Asn293Leu). Each compound had similar efficacy (tau values) on both the wild-type and mutant receptor, although tau values varied considerably among the eight compounds studied. It appeared that one derivative of isoproterenol, but not of clenbuterol, showed a gain in affinity from the wild type to the mutant receptor. This derivative had a methyl substituent instead of the usual beta-OH group in the aliphatic side chain of isoproterenol, compatible with the more lipophilic nature of the leucine side chain. Such a "gain of function" approach through a combination of synthetic chemistry with molecular biology, may be useful to enhance our insight into the precise atomic events that govern ligand-receptor interactions.

Urinary disturbance frequently develops following therapy for cervical cancer; however, no effective medical treatment has so far been reported. Sixty-five patients who developed urinary disturbance after radiation therapy, radical hysterectomy or radical hysterectomy with radiation therapy for cervical cancer underwent urodynamic assessment. Those who underwent radical hysterectomy with radiation therapy experienced the most severe urine loss, as determined by the pad test. All patients showed markedly reduced bladder compliance. A beta2-agonist (mabuterol) significantly improved compliance, bladder capacity and flow rate. It is suggested that medication with mabuterol is a potential novel approach to the treatment of urinary disturbance after therapy for cervical cancer.

A derivatization procedure for confirmatory residue analysis of beta2-agonists is described. Methyl (MBA) and butyl (BBA) boronic acids are simultaneously used for the derivatization of tulobuterol, mabuterol, mapenterol, salbutamol, clenproperol, clenbuterol, clenpenterol and bromobuterol by GC-MS determination. A temperature of 55 degrees C during 60 min was selected as optimal temperature-time condition for simultaneous MBA and BBA beta2-agonists derivatization. It was also observed that stability of boronic derivatives was maintained at -20 degrees C over a period of four days. The proposed methodology was tested in urine and it could be applied for confirmatory residue analysis of clenbuterol-like compounds.

This study investigates the influence of low dosages of beta-agonists combined with other growth promoters on screening and confirmation methods in male veal calves. Five groups of four calves were treated for 6 weeks with combinations of low dosages of beta-agonists (beta AG, clenbuterol, mabuterol and mapenterol, 0.4 microgram/kg each twice daily) in combination with 17 beta-estradiol (E2, 5 mg per animal per 14 days), methylthiouracil (MTU, 2.857 mg/kg bw twice daily) and dexamethasone (DEX, 4 mg per animal per 10 days during the last 20 days of treatment). Another group of four untreated animals served as controls. The weight and size of the thymus was reduced in the DEX group, the MTU group showed enlarged thyroids. Histologically the prostates showed vacuolar degeneration in the beta AG animals and metaplasia in the E2 group. Some vacuolization was also observed in the controls. The testis showed impaired development in all treatment groups, E2 leading to the most severe changes. DEX led to cortical atrophy of the thymus. MTU induced hyperplastic changes in the thyroid. These results indicate that comedication does not markedly affect the histological changes induced in the hormonal target tissues. For screening purposes histology of the prostate can be used as a marker for oestrogens, whereas the weight of the thymus and thyroid can be used as an indication for the use of corticosteroids and thyreostatics, respectively. Vacuolization in the prostate appeared no reliable indication for beta-agonists. Samples of urine, faeces, liver and eye (retina/choroid) were analysed for beta-agonists. E2 significantly increased the levels of all beta-agonists in the liver and faeces, whereas DEX significantly reduced these levels. These observations show that additional medication with different groups of growth promoters can markedly alter the excretion of beta-agonists and thus influence (regulatory) control.

beta-Agonists are substances used in veterinary and human medicine for the treatment of pulmonary disorders. They have found a use as growth promoters to improve meat-to-fat ratios in cattle but they are not authorized for use in the European Union. Due to their presence in trace levels (usually less than 1 microgram/kg), to the diversity of the illegally used compounds and to the complexity of the biological matrices analysed, the detection of these residues requires a very sensitive and specific method of determination. This work describes the strategy of analysis we developed for five beta-agonists in urine and liver. The combination of improved solid- or liquid-phase extraction methods and LC or GC-MS-MS (in the multiple reaction monitoring mode) has shown to provide a system suitable for the control of these substances. The efficiency of extraction and the sensitivity and selectivity allow this multiresidue detection down to, and below, the UK regulatory level of 0.5 microgram/kg. Moreover, the use of LC removes the need for the derivatisation step (cyclic methylboronate derivatives) which is required prior to GC-MS-MS analysis.

A method is described for the determination of the beta 2-agonists clenbuterol and salbutamol in hair. The method involves washing hair in sodium dodecyl hydrogensulphate solution, chemical digestion of the hair matrix in alkaline medium, solid-phase extraction, derivatization with methylboronic acid and analysis by gas chromatography/electron impact mass spectrometry in either the selected-ion monitoring or the scan mode. the effects of chemical digestion and of extraction on the recovery of the analytes were evaluated. Derivatization with methyl-boronic acid was compared with trimethylsilylation for GC/MS analysis of hair extracts, and was found to give mass spectra which showed more structural information with less chemical noise and better sensitivity. The proposed method was tested on real hair samples obtained from guinea pigs treated with growth-promoting doses of clenbuterol and salbutamol. Both compounds could be detected in hair of treated animals.

beta 2-Agonist drugs may be illegally used as growth promoters for feedlot calves, when mixed into milk replacer immediately before feeding. To check for the presence of clenbuterol, salbutamol and terbutaline in such food, an analytical system was established using a screening method based on two commercial qualitative competitive ELISA tests, with antibodies raised against the arylamino group and the t-butyl group. The extraction procedure was based on precipitation of the milk samples with acetonitrile followed by filtration. The absence of any significant interference by other substances in the filtrate allowed detection of beta 2-agonist drugs in spiked samples at the lowest concentration having a repartitioning effect (50 ppb for clenbuterol, mabuterol and terbutaline, 500 ppb for salbutamol). In view of a false positive response with tetracycline in milk samples and a cross-reaction between clenbuterol and mabuterol, an HPLC-MS technique was developed which, after extraction and purification of the samples with SPE C18 Polar Plus, was able to confirm the presence of these drugs. The good recovery after extraction (ranging from 84% to 90.2%) and the low detection limit with this method (250 ng/ml for clenbuterol, mabuterol and terbutaline, and 2.5 micrograms/ml for salbutamol) allowed easy confirmation and simultaneous detection of the four beta 2-agonists at the lowest concentrations at which they are used in adulterated milk for calves.

A method is described for the extraction of four beta-agonists, clenbuterol, salbutamol, mabuterol and terbutaline from bovine urine and liver samples using radioimmunoassay (RIA) as the method of determination. Following enzymic digestion of the liver samples using protease enzyme, the digest is centrifuged and the harvested supernatant is saturated with sodium chloride and adjusted to pH 11.0. An ethyl acetate-propan-2-ol mixture is used to extract the beta-agonists from the liver digest. The samples of urine and liver extracts are adjusted to pH 6.0 and applied to mixed phase (XtrackT) columns. The column is washed with water and methanol and the beta-agonists are eluted with methanol containing 2% ammonia. After evaporation of the eluting solvent and reconstitution in ethanol the beta-agonist residues are determined by RIA, with standard graphs prepared in residue-free sample extract. The procedure has been validated for clenbuterol, salbutamol, mabuterol and terbutaline. The mean recovery of the beta-agonists from urine and liver is > 75% and > 85%, respectively. The detection limit is 0.13 ng ml-1 and 0.46 ng g-1 of clenbuterol in urine and liver, respectively. The high recoveries attained for both types of beta-agonists are a result of an efficient liquid-liquid extraction step coupled with a selective mixed solid-phase extraction procedure.

The role of beta 2-adrenoceptor on the pathogenesis of insulin resistance in essential hypertension (EH) was explored. After the measurement of blood pressure in 15 EH patients and 8 control subjects, EH patients were divided into two groups by the elevation of plasma NE (delta NE) 5 min after standing: 7 normoadrenergic EH (delta NE < 140 pg/ml) and 8 hyperadrenergic EH (delta NE > or = 140 pg/ml). On the morning after a 12-h overnight fast, regular insulin (0.1 U/kg) was injected intravenously, and glucose disappearance rate (GDR) was measured and used as an index of insulin sensitivity. On the following day, the test was reinvestigated following the administration of mabuterol, a beta 2 agonist. Plasma growth hormone (GH), cortisol, norepinephrine (NE) and epinephrine (Epi) were measured before and after the mabuterol administration. Although there were no significant differences of basal GDR among these three groups, mabuterol induced a considerable decrease in GDR in EH patients but not in control subjects. There was no significant difference in the decrease of GDR between normo- and hyperadrenergic EH. The decrease in GDR tended to correlate with the mean blood pressure at rest in EH but not in normal subjects. Plasma glucose and serum insulin in EH patients were increased more than in normal subjects. Plasma GH, cortisol and Epi were not elevated by mabuterol, but plasma NE increased in each group, significantly in hyperadrenergic EH. There was no correlationship between the increase in plasma NE and the decrease in GDR after mabuterol.(ABSTRACT TRUNCATED AT 250 WORDS)

Two beta-adrenoceptor agonists, clenbuterol and ketoclenbuterol, were examined for their effects on growth and cardiac tissue. In female rats, clenbuterol caused a 48% increase in weight gain (P < .05), with improved feed efficiency (26%; P < .1) and increased muscle mass (9%; P < .1). Ketoclenbuterol had less effect on weight gain (30%) and feed efficiency (16%) and did not increase muscle mass. Next we studied the adverse cardiovascular effects of these compounds. Neither drug increased the force of contraction of isolated rat ventricular papillary muscle. Clenbuterol was potent at causing an increase in the rate of contraction of isolated rat atria, and when fed to cattle over 2 d, the drug caused heart rate to increase by 92 to 117%. In contrast, ketoclenbuterol was not a potent stimulator of atrial rate in the rat, and in cattle it caused a smaller increase in heart rate than clenbuterol (12 to 27%). Finally, cattle that were underfed to simulate dry-season tropical pasture conditions were treated with clenbuterol or ketoclenbuterol for 35 d. Ketoclenbuterol caused no beneficial changes in N metabolism. The results obtained with clenbuterol were equivocal, and might have been confounded partly by the refusal of some treated animals to eat all the feed offered. Although clenbuterol did not cause a reduction in total urinary N output relative to control animals, marked reductions in plasma urea concentrations and in urea synthesis were observed (23 to 53%; P < .001). We conclude that ketoclenbuterol is not effective for attenuation of dry-season protein loss in cattle. Clenbuterol seems to be less effective in underfed cattle than in well-fed cattle, and further evidence is required to judge whether compounds of this nature are likely to benefit tropical cattle under harsh grazing conditions.

To meet the requirements of the European Community for confirmatory analysis of clenbuterol using low-resolution mass spectrometry, usually two different techniques (i.e. electron impact and chemical ionization) have to be applied to confirm unambiguously its presence in extracts of urine. This paper describes the application of two different derivatives and the simultaneous analysis of these two different derivatives in one gas chromatographic-mass spectrometric analysis. With the proposed combination of techniques, Community requirements can more easily be met in only one analytical run. Examples of the analysis of some urine samples are presented, as well as data on linearity, repeatability and equivalence of the combined technique to separate determinations.

The purpose of this study was to develop a method for evaluating pulmonary clearance in mice that inhaled aerosolized 99mTc-labeled human serum albumin (33mTc-HSA). Mice were ventilated with an aerosol of 99mTc-HSA for 10 min with an ultrasonic nebulizer in an acrylic chamber. Pulmonary clearance was estimated from lung retention ratio at various times after inhalation of 99mTc-HSA. Lung retention ratios in mice were 72.4 +/- 3.3% at 1 hr, 60.1 +/- 2.3% at 3 hr and 34.8 +/- 2.0% at 24 hr after inhalation of 99mTc-HSA, similar to the previous results obtained in humans by cinescintigraphy. The data were analyzed on the basis of two-compartment model. Mabuterol, a beta 2-adrenoceptor agonist, stimulated the pulmonary clearance. In contrast, metyrapone, an adrenal 11-beta steroid hydroxylase inhibitor, suppressed the clearance with a decreased corticosterone content in plasma. These findings suggest that the developed method is useful for evaluating the pharmacological effects on pulmonary clearance.

It is well known that urinary disturbance often appears after radical hysterectomy for uterine cancer and is aggravated by additional radiation therapy. The aim of this study is to elucidate the pathogenesis and to establish the treatment method of urinary disturbance after therapy for uterine cancer. Forty-five patients with urinary disturbance and ten normal controls were subjected to this study. Changes in clinical symptoms and findings in the Urodynamic study (UDS) in 12 severe cases were investigated before and after treatment with beta 2-stimulant (Mabuterol HCL). Clinical symptoms in cases treated by radiation therapy alone were rare and mild without any pad exchanges, and appeared 5 years after treatment for uterine cancer. Findings of UDS in these cases were mild low compliance of detrusor at maximum desire to void (Cmdv) and mild low bladder volume at maximum desire to void (Vmdv). In cases treated by radical hysterectomy alone, Cmdv decreased immediatelly after the operation and then maximum urethral closure pressure (cPura) gradually decreased. Concerning the cases treated by radical hysterectomy and radiation, severe low Cmdv and severe low Vmdv appeared 5 years after the treatment for uterine cancer in almost all cases, and low cPura appeared immediately after the operation in half of the cases. Treatment with beta 2-stimulant significantly improved urinary frequency, voided volume and urinary incontinence. In UDS findings, Vmdv and Cmdv were significantly improved by the treatment with beta 2-stimulant. The functional profile length and cPura value were not significantly changed. In uroflowmetry, the maximum flow rate and average flow rate were significantly improved by the treatment with beta 2-stimulant.(ABSTRACT TRUNCATED AT 250 WORDS)

The longissimus dorsi muscles of cattle are highly responsive to the anabolic effects of beta 2-adrenoceptor agonists, and in the present study were shown to be a rich and homogeneous source of beta 2-adrenoceptors. Structural analogues of the beta 2-adrenoceptor agonist clenbuterol were prepared in order to examine the relative importance of the benzylic hydroxyl functionality and the aromatic ring halogen substituents in determining the affinity of phenylethanolamines for beta 2-adrenoceptors in bovine muscle. It was calculated that the hydroxyl-hydrogen bonding interaction of these compounds contributes only 1-2 kcal/mol to the total binding energy. The aromatic halo-substituents contributed up to 3 kcal/mol to the total binding energy, and we suggest that the relative importance of the latter functionality has been previously underestimated. There was a poor correlation between the affinity of clenbuterol analogues for binding to beta 2-adrenoceptors, and the potency of these compounds in reducing urinary nitrogen excretion after oral administration to female rats. We suggest that beta 2-adrenoceptor agonist efficacy is reduced in phenylethanolamine compounds when iodine is present on the aromatic ring. In contrast, the increased potency of a ketone derivative might be explained by conversion in vivo to clenbuterol, with increased bioavailability of this beta 2-agonist at the site of action.

We studied the effect of mabuterol on the tracheal mucociliary clearance of magnetized iron particles in anesthetized dogs. A catheter was inserted directly into the trachea through the mouth and 0.05 ml of saline solution with 30 mg of Fe3O4 was instilled into the trachea. After magnetization from outside the chest wall, the remanent magnetic field (RMF) strength generated in the trachea was sequentially measured with a flux-gate magnetometer. The decay of RMF immediately after sequential magnetization showed a clearance of Fe3O4 particles. Mabuterol (10 micrograms/kg) increased clearance as much as isoproterenol (10 micrograms/kg) 30 min after intravenous injection relative to control measurement (p less than 0.01). These results suggest that mabuterol is useful to promote clearance.

We examined the inhibitory effect of the long-acting beta-adrenergic agonists, mabuterol, clenbuterol and fenoterol on "morning dipping" in ten patients with nocturnal asthma. On the first night, as a control experiment, the subjects received no beta-adrenergic agonist. On the succeeding three nights at 8:00 PM, each subject was orally administered 50 micrograms of mabuterol, 40 micrograms of clenbuterol and 5 mg of fenoterol in a randomized, crossover fashion. Pulmonary function tests (FVC, FEV1.0, PEFR, V50 and V25) were performed at 8:00 PM (just before administration of beta-adrenergic agonist), 9:00 PM, 10:00 PM, 6:00 AM and 8:00 AM. On the night when clenbuterol was administered, there was a significant inhibition of morning dipping at 6:00 AM in FVC (p less than 0.01), FEV1.0 (p less than 0.01), PEFR (p less than 0.01), V50 (p less than 0.01) and V25 (p less than 0.05) compared with the control night. On the nights when mabuterol and fenoterol were administered, there was a significant inhibition of morning dipping at 6:00 AM in FVC (p less than 0.01) and FEV1.0 (p less than 0.01) compared with the control night. Palpitations associated with clenbuterol administration were seen in two subjects. The effect of each beta-adrenergic agonist varied inconsistently among the subjects. These results indicate that clenbuterol is the most effective in inhibiting morning dipping among the long-acting beta-adrenergic agonists examined, but individualization in the choice of beta-adrenergic agonist is mandatory in order to achieve the maximum effect.

Effects of dl-1-(4-amino-3-chloro-5-trifluorome-thyl-phenyl)-2-tert.-bu tylamino-ethanol hydrochloride (mabuterol) on the cardiovascular system and smooth muscle organs were investigated in comparison with those of isoprenaline (isoproterenol), salbutamol and procaterol, and the following results were obtained. Mabuterol (i.v.) produced a dose-dependent decrease in the blood pressure at doses ranging from 0.3-1000 micrograms/kg, and the heart rate was slightly increased at 0.3-30 micrograms/kg but decreased at 100-1000 micrograms/kg in rats, cats and dogs. The effects of mabuterol on the blood pressure, total peripheral vascular resistance (TPR) and heart rate were respectively 14, 37 and 104 times less potent than those of isoprenaline and there were none on the maximum dp/dt in the left ventricle in cats. In dogs, mabuterol had 356 and 118 times less effect on the blood pressure and TPR than isoprenaline and slightly increased the heart rate and maximum dp/dt. Mabuterol (p.o.) decreased the blood pressure at doses ranging from 3 micrograms-100 mg/kg, and the heart rate was increased at 3 micrograms-10 mg/kg, but decreased at 100 mg/kg in conscious and anesthetized rats. In the guinea pig atria, mabuterol slightly increased the heart rate and contractile force, but in the electrically driven left atrium mabuterol had no effect whereas procaterol slightly increased the contractile force. Mabuterol inhibited the positive inotropic effect of isoprenaline at 10(-7) g/ml and decreased the maximum driving frequency at 3 X 10(-6) g/ml. Mabuterol was 3 times more potent in relaxing the isolated rat uterus, but 700 times less potent than isoprenaline in relaxing the rabbit jejunum. Mabuterol (p.o.) depressed the intestinal propulsion and was equipotent to isoprenaline and 2.5 times less potent than salbutamol. Mabuterol had no effect on alpha-adrenergic, acetylcholine and histamine receptors. These results suggest that mabuterol has a specific effect on beta 2-adrenoceptors with no beta 1-stimulation. In addition, mabuterol exhibited beta 1-blocking activity at dosages 200-300 times higher.

Pharmacokinetic studies on absorption, distribution, excretion and placental transfer of the beta-sympathomimetic 14C-labelled dl - 1 - (4 - amino - 3 - chloro - 5 - trifluoromethyl-phenyl)-2-tert.-butylamino-ethanol hydrochloride (mabuterol) have been carried out in rats. Mabuterol was easily absorbed from the whole length of the small intestine in a study in situ with the gastro-intestinal ligated loop technique. The blood and tissue levels of radioactivity reached the maximum within 1 h after oral administration. Concerning tissue distribution (excluding the digestive tract) after oral administration, the radioactivity in the liver, lung, kidney and several secretory organs was considerably higher than that in the blood. The levels of radioactivity in these tissues continued at almost the same levels for 2-6 h after oral administration, resulting from a slow gastric emptying of the radioactivity. The whole-body autoradiograms obtained after intravenous administration revealed the secretion of the radioactivity into the stomach. The radioactive concentration in the CNS was much lower than that in the principal organs with either route of administration. About 60% and 26% were excreted into urine and feces, respectively, within 24 h after oral administration. Although about 22% was excreted into bile within 24 h after intravenous administration, no other evidence of enterohepatic circulation was observed. The multiple oral administration of 14C-mabuterol indicated no, appreciable accumulation in the tissues of rats. A placental transfer in the pregnant rats in the last stage of gestation was not significant.