Metaproterenol, isoproterenol, and their bisdimethylcarbamate derivatives as human cholinesterase inhibitors.

Metaproterenol and isoproterenol are bronchodilators that provide a structural basis for many other bronchodilators currently in use. One of these structurally related bronchodilators is terbutaline; it is administered as a prodrug, bambuterol, and is metabolized (bioconverted) into terbutaline by butyrylcholinesterase (BChE). The metabolism rate can be affected by BChE gene polymorphism in the human population and BChE stereoselectivity. The aim of our study was to investigate inhibition of human BChE and acetylcholinesterase (AChE) with metaproterenol, isoproterenol, and newly synthesized racemic bisdimethylcarbamate derivatives of metaproterenol (metacarb) and isoproterenol (isocarb) and their (R)-enantiomers to see if their bioconversion is affected by BChE inhibition in the same way as that for bambuterol. Metacarb and isocarb proved to be selective BChE inhibitors, as they progressively inhibited AChE 960 to 80 times more slowly than BChE(UU). All studied cholinesterases displayed poor affinity for metaproterenol and isoproterenol, yet BChE(UU) had an affinity about five times higher than that of AChE.

Beta2-adrenoceptor agonists for dysmenorrhoea.

BACKGROUND: Dysmenorrhoea is a common gynaecological complaint that can affect as many as 50% of premenopausal women, 10% of whom suffer severely enough to be rendered incapacitated for one to three days during each menstrual cycle. Primary dysmenorrhoea is where women suffer from menstrual pain but lack any pathology in their pelvic anatomy. Beta2-adrenoceptor agonists have been used in the treatment of women with primary dysmenorrhoea but their effects are unclear. OBJECTIVES: To determine the effectiveness and safety of beta2-adrenoceptor agonists in the treatment of primary dysmenorrhoea. SEARCH METHODS: We searched the Cochrane Menstrual Disorders and Subfertility Group Specialised Register; CENTRAL (The Cochrane Library 2011, Issue 8); MEDLINE; EMBASE; PsycINFO and the EBM Reviews databases. The last search was on 22 August 2011. SELECTION CRITERIA: Randomised controlled trials comparing beta2-adrenoceptor agonists with placebo or no treatment, each other or any other conventional treatment in women of reproductive age with primary dysmenorrhoea. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed trial quality and extracted the data. MAIN RESULTS: Five trials involving 187 women with an age range of 15 to 40 years were included. Oral isoxsuprine was compared with placebo in two trials; terbutaline oral spray, ritodrine chloride and oral hydroxyphenyl-orciprenalin were compared with placebo in a further three trials. Clinical diversity in the studies in terms of the interventions being evaluated, assessments at different time points and the use of different assessment tools mitigated against pooling of outcome data across studies in order to provide a summary estimate of effect for any of the comparisons. Only one study, with unclear risk of bias, reported pain relief with a combination of isoxsuprine, acetaminophen and caffeine. None of the other studies reported any significant clinical difference in effectiveness between the intervention and placebo. Adverse effects were reported with all of these medications in up to a quarter of the total number of participants. They included nausea, vomiting, dizziness, quivering, tremor and palpitations. AUTHORS' CONCLUSIONS: The evidence presented in this review was based on a few relatively small-sized studies that were categorised to have unclear to high risk of bias, which does not allow confident decision-making at present about the use of beta2-adrenoceptor agonists for dysmenorrhoea. The benefits as reported in one study should be balanced against the wide array of unacceptable side effects documented with this class of medication. We have emphasised the lack of precision and limitations in the reported data where appropriate.

Cromoglycate, reproterol, or both--what's best for exercise-induced asthma?

OBJECTIVE: International guidelines recommend short- (SABA) or long-acting b-agonists for the prevention of bronchoconstriction after exercise (EIB) in patients with exercise-induced asthma (EIA). However, other drugs are still in discussion for the prevention of EIB. We investigated the efficacy of a combination of inhaled sodium cromoglycate and the ß-mimetic drug reproterol versus inhaled reproterol alone and both versus inhaled placebo in subjects with exercise-induced asthma (EIA). METHODS: The study aimed to prove the preventive effect of a combination of 1-mg reproterol and 2-mg disodium cromoglycate (DSCG) and its single components vs. placebo, measuring the decrease of FEV1 after a standardized treadmill test in 11 patients with recorded EIA. The study medication was twice as high as those of drugs which are commercially available (e.g., Allergospasmin®, Aarane®). RESULTS: The results revealed that the combination of reproterol and DSCG was significantly effective against a decrease of FEV1 after a standardized exercise challenge test (ECT) compared to placebo. The short-acting b-agonist reproterol alone had almost the same effectiveness as the combination of reproterol and DNCG. The difference between the combination with DNCG and reproterol alone was less than 10% and insignificant (p 0.48). DNCG alone did not show a difference in the effectiveness compared to placebo. CONCLUSION: Prevention of EIA with the combination of reproterol and DSCG or with reproterol only is effective. An exclusive recommendation in favor of the combination cannot be given due to the low difference in the effectiveness versus reproterol alone. Due to the limited number of subjects and some probands showing protection under DSCG, it cannot be completely excluded that there is some preventive power of DSCG in individual cases.

Prevention of exercise-induced asthma by a fixed combination of disodium cromoglycate plus reproterol compared with montelukast in young patients.

BACKGROUND: The leukotriene inhibitor montelukast has been recommended against exercise-induced asthma (EIA), however, single-dose agents might be favourable in several aspects. OBJECTIVE: To compare the protective effects against EIA of a single inhalation of the combination disodium cromoglycate (DSCG, CAS 16110-51-3) and reproterol (REP, CAS 54063-54-6) with 3 days oral treatment of montelukast (MON, CAS 158966-92-8). METHODS: Open-label, cross-over, single-centre trial. Twenty-four 6 to 18-year-old children and adolescents, with reversible and stable airway obstruction, baseline FEV1 > or = 70%, predicted and proven EIA (i.e. a maximum decrease of FEV1 by > or = 20% compared with baseline) were treated with MON, orally for 3 days in the evening, or one single inhalation of DSCG/REP 20 min before the exercise challenge. The treatment sequence was randomised. The exercise test on a treadmill was performed under standardised conditions. RESULTS: 24 patients completed both periods. Both treatments clearly provided protection against EIA; however, protection of DSCG/REP was more pronounced than that of MON. This difference was statistically significant even if the data were adjusted for the increase in FEV1 between inhalation of DSCG/REP and challenge (DSCG/REP(adjusted). The nadir FEV1 level after exercise following prophylaxis with DSCG/REP was even higher than the pre-inhalation FEV1 value. From these data, protection indices of 66%, 81%, and 113% for MON, DSCG/REP(adjusted), and DSCG/REP(unadjusted), respectively, were estimated. CONCLUSIONS: Inhalation of DSCG/REP before exercise provides significantly better protection against EIA than three days treatment with MON.

Multiresidue analysis of beta-agonists in bovine and porcine urine, feed and hair using liquid chromatography electrospray ionisation tandem mass spectrometry.

The use of beta-agonists as growth promoters in cattle breeding is forbidden in many countries for reasons of fair trade and consumer protection. In recent years the use of liquid chromatography (LC) tandem mass spectrometry (MS/MS) has been shown to be the method of choice for the control of beta-agonists. In this study an LC-MS/MS multiresidue analysis method is presented for trace analysis of 22 beta-agonists. A truly generic concept has been designed based on mixed-mode solid-phase extraction and positive electrospray ionisation LC-MS/MS operated in the multiple reaction monitoring mode. This method allows application to a wide variety of sample matrices such as urine, feed and hair, following minor modifications to the analysis procedure only. The method features fit-for-purpose sensitivity in urine as shown by CCalpha and CCbeta values of less than 0.2 and less than 0.5 microg/l respectively, for all beta-agonists studied (terbutaline and reproterol, less than 0.3 and less than 1.0 respectively). Similar but semiquantitative application to feed and hair showed CCbeta values of less than 10.0 and less than 5.0 microg/kg, respectively. A further simplification and improvement is demonstrated using Ultra Performance LC (UPLC) and fast-switching MS/MS. The successful validation of this method following the latest EU requirements and its application to real samples demonstrate that a new versatile tool has been achieved for veterinary control of beta-agonists.

Mononuclear cell membranes: stabilization by reproterol and cromoglycate, destabilization by fenoterol and salbutamol.

Electron paramagnetic resonance (EPR) spectroscopy with spin labels 5- and 16-doxyl-stearic acid (DSA) was used to differentiate between actions of beta-agonists on human mononuclear cell membrane. Reproterol (CAS 13055-82-8), salbutamol (CAS 51022-70-9) and fenoterol (CAS 1944-12-3) compared to cromoglycate (CAS 15826-37-6) were used at concentrations of 10-100 nmol/l per 10(7) cells. With reproterol, order and polarity was not much changed, whereas salbutamol and fenoterol significantly destabilized the membrane to similar extent. Cromoglycate acted in a stabilizing fashion. With trypan blue exclusion, reproterol and cromoglycate showed stable values, whereas salbutamol and fenoterol augmented permeability. Thus, by conventional lipid spin labeling the discrimination between salbutamol and fenoterol could not be carried out. In contrast, previous lipid peroxidation studies in a model system had revealed a decrease by reproterol, no change by salbutamol and an increase by fenoterol. Also, using fenoterol, protein spin label 4-maleimido-TEMPO (2, 2, 6, 6-tetramethyl-1-piperidinyloxy) showed an increase of membrane rigidity of mononuclear cells. Moreover, mast cells of different origin were previously found tween beta-agonists. Reproterol in all tests behaved in a therapeutically profitable way. In conclusion, in addition to lipid spin labeling other methods and materials should be considered, to finally arrive at a more realistic differentiation between, for instance, salbutamol and fenoterol. The term "membrane (de) stabilization" should not generally be used without careful consideration of the type of cell/membrane in question.

Effect of reproterol either alone or combined with disodium cromoglycate on airway responsiveness to methacholine.

Regular use of inhaled beta2-agonists might lead to tolerance as reflected in a loss of bronchoprotection. In vitro-data suggest that this might be prevented by disodium cromoglycate (DSCG). Therefore, we studied the effect of the beta2-agonist reproterol in combination with DSCG. In a cross-over design, 19 subjects with airway hyperresponsiveness inhaled either placebo, 1mg reproterol, 2 mg DSCG, or 1mg reproterol plus 2 mg DSCG 4x daily over 2 weeks. Treatment periods were separated by > or = 7 days. Before and at the end of periods, lung function and methacholine responsiveness were determined in the morning, and 6h later the bronchodilator effect and the protection against methacholine-induced bronchoconstriction. Reproterol or DSCG or their combination did not exert detrimental effects on lung function, airway responsiveness, or bronchodilator capacity. However, bronchoprotection was significantly reduced (p < 0.05) after treatment with placebo, reproterol or reproterol plus DSCG, the respective changes being 0.59, 0.96 and 1.37 doubling concentrations. All changes were small as compared to intraindividual variability. In this model all treatments except with DSCG caused a significant but small loss of protection against methacholine-induced bronchoconstriction. Thus, tolerance was not prevented by 2 weeks of additional treatment with DSCG, in contrast to in vitro findings.

Different mechanisms of action of beta2-adrenergic receptor agonists: a comparison of reproterol, fenoterol and salbutamol on monocyte cyclic-AMP and leukotriene B4 production in vitro.

BACKGROUND: Beta2-adrenergic receptor agonists have several effects on airway function, most of which are mediated in a variety of cell types resulting in increased c-AMP-production and inhibition of inflammatory mediator production. However, their stimulating effects on cAMP-production became known to be inversed by increasing phosphodiesterase (PDE) activity and degradation of cAMP. Therefore, in this study we have evaluated the efficacy of reproterol, a dual acting beta2-adrenoceptor agonist and PDE-inhibitor, as compared to salbutamol and fenoterol with respect to production of cAMP and LTB4 in cultured monocytes. METHODS: Isolated human monocytes (10(5)/ml) were incubated (n = 9) in suspension with beta2-adrenoceptor agonists (10(-10) -10(-4) M) for 30 minutes with and without IBMX. Then, cAMP production was determined following treatment with Triton-X100. Production of LTB4 was measured following incubation of beta2-adrenoceptor agonists for 4 hrs in the presence of LPS (10 mg/ml). cAMP and LTB subset 4 were measured in culture supernatants by enzyme immunoassay. RESULTS: At 10(-5) M, production of cAMP was significantly stimulated by reproterol > fenoterol > salbutamol in a dose-dependent manner to an extent of *128%, *65%, 13% (*p<0.04) respectively. In contrast, LTB4-production was inhibited significantly to a similar degree by salbutamol and reproterol in a dose-dependent manner by 59% and 49% (10(-5) M, p<0.03), respectively, with decreasing inhibition (15%) after fenoterol. Following co-incubation with IBMX, cAMP production only increased significantly (p<0.002) after fenoterol (+110%) compared to salbutamol (+29%) and reproterol (+50%) (ANOVA, p<0.001). CONCLUSION: These data suggest effects of the theophylline constituent of reproterol to inhibit adenylyl cyclase induced phosphodiesterase activity. The advantageous synergistic effects of reproterol on cAMP-production need to be further explored in trials.

Inhibition by reproterol of cAMP PDE in intact mastocytoma P-815 cells.

In vitro studies in rat mastocytes and human monocytes suggested that reproterol (a selective beta(2)-adrenoceptor agonist with a theophylline moiety) exerts anti-inflammatory actions through inhibition of cyclic AMP (cAMP) PDE activity. Thus, reproterol was tested for its ability to inhibit cAMP PDE in cultured mouse mastocytoma P-815 cells. cAMP PDE activity was measured in intact cells by spectrofluorometry using the fluorescent substrate 2'-O-anthraniloyl cAMP. Reproterol was more potent than theophylline to inhibit cAMP PDE (pIC(50)=4.28+/-0.25 vs. 3.16+/-0.05). This contrasted with disrupted cells, where the PDE inhibitory potency of reproterol was low (pIC(50)=2.85+/-0.03) and similar to that of theophylline (pIC(50)=2.66+/-0.19). No cAMP PDE inhibition was found with other beta(2)-agonists tested (fenoterol, salbutamol, salmeterol and formoterol). Finally, the selective PDE inhibitors calmidazolium (100 nM), milrinone (5 microM) and rolipram (50 microM) inhibited cAMP PDE activity by approximately 20, 30 and 25% respectively. In conclusion, reproterol potently and non-specifically inhibited intracellular cAMP phosphodiesterases in intact mastocytoma cells. This can explain the previously reported beta(2)-adrenoceptor-independent anti-inflammatory actions of reproterol in vitro. Further studies are required to define the anti-inflammatory potential of reproterol in asthma.

Conductimetric determination of reproterol HCl and pipazethate HCl and salbutamol sulphate in their pharmaceutical formulations.

A simple and sensitive conductimetric method for the determination of salbutamol sulphate and reproterol and pipazethate hydrochlorides is presented based on their ion associates with phosphotungstic and phosphomolybdic acids. The effect of solvent, molar ratio, reagent concentration and temperature were studied, and the solubility products of the formed ion associates were calculated. The method was applied to the determination of the drugs in their pure state or pharmaceutical preparations with mean recovery values of 99.82-100.54, 99.75-100.12 and 99.95-100.40%, and coefficient of variation 0.28-0.52, 0.16-0.36 and 0.19-0.33 for salbutamol sulphate, reproterol HCl and pipazathate HCl, respectively.

Screening of beta-2 agonists and confirmation of fenoterol, orciprenaline, reproterol and terbutaline with gas chromatography-mass spectrometry as tetrahydroisoquinoline derivatives.

A new method for a comprehensive screening and confirmation of beta-2 agonists in human urine is presented based on gas chromatography-low-resolution mass spectrometry (GC-MS) using electron impact ionisation (EI). After hydrolysis of the conjugates with beta-glucuronidase/arylsulfatase a derivatisation step with formaldehyde converts fenoterol, orciprenaline, reproterol and terbutaline to one derivative, a tetrahydroisoquinoline, while the other beta-2 agonists remain unchanged. Liquid-liquid extraction and trimethylsilylation follow. The tetrahydroisoquinoline derivatives show good gas chromatographic and mass spectrometric behaviour. The detection limit of these four beta-2 agonists in the screening using low-resolution mass spectrometry is 10 ng/ml of urine. The other beta-2 agonists are detected as parent compounds with the same recovery after sample preparation with and without formaldehyde. The EI mass spectra of the tetrahydroisoquinoline derivatives are presented.

beta-agonistic bronchodilators: comparison of dose/response in working rat hearts.

STUDY OBJECTIVES: Different beta-agonists are compared with regard to their cardiodepressive side effects. DESIGN: The metaphenolic bronchodilators reproterol, salbutamol, fenoterol, and terbutaline were introduced at a dosage of 0.0005 micromol to a maximum of 10 micromol per gram of heart tissue into the isolated working rat heart under hypoxic conditions, and the response was observed during subsequent reoxygenation. As an index of external heart work, aortic flow was measured. Heart rate, coronary flow, and developed pressure were recorded. At the end of heart perfusion, mitochondria were isolated and analyzed for adenosine triphosphatase activity, adenosine triphosphate (ATP) synthesis, and membrane fluidity. Moreover, intact mitochondria and lipid peroxidation were investigated using a model system. MEASUREMENTS AND RESULTS: Compared to controls, reproterol gave the most favorable results, with an increase of 25 to 30% of aortic flow during reoxygenation at a concentration of 10 micromol/g heart tissue. In contrast, both fenoterol and salbutamol at a concentration of 1 micromol/g heart tissue decreased aortic flow during reoxygenation, whereas terbutaline had a negative influence on aortic flow at 0.01 to 0.1 micromol/g heart tissue. Mitochondria of these hearts were isolated at the end of the experiment. Mitochondrial ATP synthesis was increased above controls at nearly all concentrations of reproterol. ATP synthesis was decreased at 1 micromol and 10 micromol fenoterol. As little as 0.0005 micromol terbutaline decreased ATP synthesis by 50%. In intact mitochondria, adenosine diphosphate (ADP) to oxygen ratios were found to be increased with terbutaline and fenoterol, indicating ADP consumption by myokinase activation. Lipid peroxidation was increased in a model system between concentrations of 0.002 micromol/mg and 0.04 micromol/mg phosphatidylcholine by fenoterol and terbutaline, whereas a decrease was noted with reproterol. Membrane fluidity was found increased after addition of reproterol, which supports the evidence of efficient ATP synthesis by this compound. CONCLUSIONS: Cardiodepressive side effects and greater toxicity of fenoterol and terbutaline were found under the conditions of our experiment. Salbutamol and, in particular, reproterol appear much better tolerated. In addition to partial beta-adrenergic agonism, reproterol may exert an inhibitory influence on adenosine receptor sites and phosphodiesterase, which could result in membrane stabilization by saving cyclic adenosine monophosphate or ATP.

Reproterol--A monomolecular combination of orciprenaline and theophylline: novel aspects of its mode of action in asthma.

BACKGROUND AND OBJECTIVE: Reproterol is a monomolecular combination of orciprenaline and theophylline used as beta-adrenergic agonist to induce bronchodilation in bronchial asthma. Since the mechanism of action of reproterol has not been investigated so far, its potential anti-inflammatory activity in asthma remains still unknown. Therefore, we have studied in vitro whether the theophylline component of the reproterol molecule might enhance the stimulatory effect of the beta-adrenoceptor on cAMP production resulting in suppression of inflammatory mediator production. METHODS: The effects of reproterol, orciprenaline and theophylline (10(-9)-10(-5) M) on spontaneous cAMP (5 x 10(4) cells/30 min)- and on LPS (10 microg/ml)-stimulated LTB4 production (10(5) cells/4 h) were determined in normal monocytes in vitro. RESULTS: Production of cAMP (n = 9) was significantly augmented in a dose-dependent manner by orciprenaline (30 +/- 8%) and theophylline (28 +/- 10%), but mostly by reproterol (127 +/- 8%) at 10(-5) M. Despite incubation with propranolol, significant stimulation of cAMP production was notable following reproterol therapy. Production of LTB4 was significantly inhibited by reproterol (-48 +/- 14%) and less by theophylline (-28 +/- 10%), but was stimulated by orciprenaline (+20 +/- 8%) at 10(-5) M. CONCLUSION: We conclude that reproterol exerts a strong stimulatory effect on monocyte cAMP production and a suppressive effect on LTB4 production possibly due to a synergistic mode of action on adenylate cyclase activity and inhibition of phosphodiesterases. More clinical studies in bronchial asthma will be needed to determine whether these results may translate into clinically relevant effects.

Excretion study of the beta2-agonist reproterol in human urine.

An excretion study of the beta2-agonist 7-[3-[(beta-3,5-trihydroxyphenethyl)amino]-propyl]theophylline (reproterol) in human urine, which is reportedly misused by athletes and horses as a doping agent, is presented. The study was performed after an oral administration of 20 mg of reproterol hydrochloride. The collected urine samples were prepared using the standard anabolic steroid extraction procedure and analyzed by gas chromatography coupled with quadrupole mass spectrometry and, also, with high-resolution mass spectrometry (HRMS). The main reproterol metabolite was found, whereas unchanged reproterol was not detected. The structure of the main metabolite was confirmed by an accurate HRMS measurement of diagnostic ions. Finally, an excretion urine profile of the main metabolite is presented. The mass spectrum of another possible unidentified reproterol metabolite is also reported.

Enhancement by reproterol of the ability of disodium cromoglycate to stabilize rat mastocytes.

The beta2-adrenoceptor agonist reproterol and disodium cromoglycate (DSCG) are used in fixed combination for the treatment of asthma, because they act on bronchial smooth muscle and inflammatory cells, respectively. Here, we investigated if reproterol can also act in rat mast cells in vitro to facilitate the inhibitory action of disodium cromoglycate (DSCG) on histamine secretion induced by compound 48/80. Reproterol was as potent as DSCG to inhibit histamine release in rat mast cells (32.8+/-6.0 vs. 36.7+/-6.2% at 1 microM of each compound, n=10 and n=8 respectively). Mast cell stabilization by DSCG (1-100 microM) was strongly and significantly enhanced in the presence of a fixed saturating concentration of reproterol (100 microM). Conversely, the combination of DSCG (1-100 microM) with the beta2-agonist used as reference compound, salbutamol (100 microM) did not inhibit histamine release more than DSCG alone. In combination with a saturating concentration of DSCG (100 microM), reproterol inhibited histamine release more than reproterol alone. The potent adenylate cyclase stimulator forskolin (50 microM) was able to inhibit histamine release to a similar extent as DSCG and significantly (P<0.05) enhanced the inhibition of histamine release by DSCG. Finally, the phosphodiesterase inhibitor theophylline (100 microM) was equipotent to reproterol and DSCG in stabilizing rat mast cells. In conclusion, reproterol enhances the ability of disodium cromoglycate to stabilize rat mast cells. This effect is not shared by salbutamol and can be, at least in part, independent of beta2-adrenoceptor stimulation.

Superoxide radical scavenging by phenolic bronchodilators under aprotic and aqueous conditions.

Asthmatic airway disease is accompanied by the appearance of inflammatory cells which produce reactive oxygen species (ROS). Therefore, the radical scavenging properties of the bronchodilators reproterol, fenoterol, salbutamol and terbutaline toward superoxide anion radicals and hydroperoxyl radicals were investigated in a model system by electron paramagnetic resonance spectroscopy (EPR) and photometric approaches. The substances under study showed activity in superoxide radical scavenging under aprotic and protic conditions as well. The efficiency of the reaction decreased in the order: fenoterol > salbutamol > reproterol > terbutaline > oxyfedrine when DMSO was used as an aprotic solvent. In an aqueous system, the rate constants decreased in the order: fenoterol > reproterol > salbutamol. It is suggested that the antioxidant effect of these beta2-agonists is an additional advantage in treatment of asthmatic lung disease, reducing the negative consequences of airway inflammation.