A new simple cell-based homogeneous time-resolved fluorescence QRET technique for receptor-ligand interaction screening.

In this article, a single-label separation-free fluorescence technique is presented as a potential screening method for cell-based receptor antagonists and agonists.The time-resolved fluorescence technique, quenching resonance energy transfer (QRET), relies on a single-labeled binding partner in combination with a soluble quencher. The quencher efficiently suppresses the luminescence of the unbound labeled ligand, whereas the luminescence of the bound fraction is not affected. This approach allows the development of cell-based screening assays in a simple and cost-effective manner. The authors have applied the technique to the screening of beta(2)-adrenoreceptor (beta(2)AR) antagonists and agonists in intact human embryonic kidney HEK293(i) cells overexpressing human beta(2)-adrenergic receptors. Two antagonists (propranolol, alprenolol) and 2 agonists (metaproterenol, terbutaline) for beta(2)AR were investigated in a displacement assay using europium(III)-labeled pindolol ligand. The assay Z' values ranged from 0.68 to 0.78, the coefficient of variation was less than 10%, and the K(i) values were 19 nM for propranolol and alprenolol and 14 and 5.9 microM for metaproterenol and terbutaline, respectively. The QRET technique with beta(2)AR was also applied to LOPAC compound library screening, yielding nearly error-free recognition of known binders. This simple and cost-effective technique can be readily adapted to laboratory and industrial-scale screening.

Chronic beta-adrenoreceptor stimulation in vivo decreased Bcl-2 and increased Bax expression but did not activate apoptotic pathways in mouse heart.

Prolonged activation of the sympathetic nervous system is deleterious to heart function. In vitro beta1-adrenergic activation promotes apoptosis, whereas beta2-adrenergic activation reduces apoptosis in cultured adult cardiomyocytes. To determine the effect of chronic catecholamine infusion in vivo, we measured apoptosis marker expression in C57Bl/6 and catecholamine-sensitive Egr-1 deficient mice after treatment with the nonspecific beta-adrenergic agonist, isoproterenol, the beta1-specific agonist, dobutamine, or the beta2-specific agonist, metaproterenol. Antiapoptotic and proapoptotic protein expression, cytochrome c release and caspases 3, 9, and 12 activation products were measured on immunoblots. Catecholamine-treated mice had decreased Bcl-2 and increased Bax and BNIP1 expression, suggesting mitochondria-dependent apoptosis pathway activation. However, cytosolic cytochrome c or caspase 3 or 9 activation products were not detected. In mice, increased molecular chaperone expression and caspase 12 activation characterize endoplasmic-reticulum-driven apoptosis. Clusterin expression was increased in catecholamine-treated mice, but GRP78 expression was not increased, and caspase 12 activation products were not detected. Thus, neither the mitochondrial nor the endoplasmic apoptotic pathway was fully activated. Further, Egr-1 deficiency did not increase cardiac apoptosis. We conclude that although chronic in vivo infusion of beta1- or beta2-adrenergic receptor agonists partially activates the apoptosis program, full activation of the caspase cascade requires more, or other, cardiac insults.

Tolerability and in vivo performance of a novel freon-free metered dose inhaler for a fixed combinational product of reproterol and disodium cromoglycate.

The present study was conducted to describe and compare the in vivo performance (systemic exposure), clinical and laboratory safety of a fixed combinational product of inhaled reproterol (CAS 54063-54-6) plus disodium cromoglycate (DSCG; CAS 15826-37-6) using a novel freon (CFC)-free metered dose inhaler (MDI), which uses 1,1,1,2,3,3,3-heptafluoropropane (HFA-227; CAS 431-89-0) as propellant and polyoxyethylene glyceryl trioleate (Tagat TO; CAS 68958-64-5) as surfactant relative to the conventional freon-driven MDI Allergospasmin in healthy male and female volunteers. Twenty-four young male and female healthy subjects were randomly allocated in gender-balanced fashion to 4 parallel treatment groups with single and repeated dosing of either reproterol + DSCG by HFA- or CFC-MDI (each time N = 8) or placebo by HFA- or CFC-MDI (each time N = 4) using matched placebo devices thus allowing a double-blind (with regard to placebo) approach. Treatments consisted of a single morning dose of 2 actuations followed 4 days later by a 1 week treatment course of 2 actuations four times daily. Subjects were investigated extensively in terms of blood pressure, pulse rate, electrocardiography, spirometry, respiratory rate, body temperature, laboratory safety (haematology, clinical chemistry, urinalysis) and clinical well-being. There were no treatment, compound or device related effects for any of the tolerability and safety end points. The treatments were well tolerated. In particular, there was no irritative cough or any sign of broncho-irritation on application. Adverse events were reported in a total of 9 subjects: 3/8, 4/8, 0/4 and 2/4 subjects treated with reproterol + DSCG by HFA-MDI, reproterol + DSCG by CFC-MDI, placebo by HFA-MDI and placebo by CFC-MDI, respectively. Of these, 6 events in 6 subjects receiving the active treatments were considered probably or definitely related to the test drug administration (i.e. adverse drug reactions): after reproterol + DSCG one subject in each treatment group (HFA-MDI and CFC-MDI) complained of an unpleasant bitter taste immediately after application; one further subject in each group complained of headache. Under treatment with reproterol + DSCG by CFC-MDI one male subject complained of mild transient nausea with onset on day 5. Under treatment with reproterol + DSCG by HFA-MDI one female subject complained of mild dizziness and mildly disturbed (blurred) vision with onset on day 1. All adverse events occurred only transitory and required no treatment. Systemic exposure, evaluated by the plasma concentrations of DSCG at 1 h after application, was slightly higher with the HFA-MDI compared to the CFC-MDI. It is concluded that the safety, tolerability and in vivo performance of the newly developed freon-free MDI is at least as well tolerable as the already marketed freon-driven conventional formulation.

Rapid and automated determination of the beta2-agonist reproterol in human plasma by atmospheric pressure chemical ionisation high-performance liquid chromatography-tandem mass spectrometry.

An on-line HPLC assay with tandem mass spectrometric detection for the fast and sensitive determination of reproterol in human plasma was developed, utilising a methylated structural analogue as the internal standard. Automated solid-phase extraction of diluted plasma samples, based on 250 microl plasma aliquots, at pH 6.5, allowed a reliable quantification of reproterol down to 400 pg/ml. Injection of 100 microl of plasma extracts onto a 30 mm x 4.6 mm reversed-phase guard column provided retention times ranging from 20 to 30 s for reproterol and the internal standard. The standard curves were linear from 0.2 to 200 ng/ml using weighted linear regression analysis (1/y2). The inter-assay and intra-assay accuracies were -0.9% and +3.2%, exhibiting a precision (C.V.) of +/- 11% and +/- 9.3%, respectively. Up to 100 unknowns may be analysed each 24 h per analyst.

Evaluation of the effect of different fatty acids on the percutaneous absorption of metaproterenol sulfate.

The in vitro permeation rates of metaproterenol sulfate (MPS) across hairless mouse skin and TESTSKIN living skin equivalent were very low unless skin permeation enhancers were included in the vehicle. An optimum balance should be established between the chain length of the fatty acid and its molar ratio to MPS in order to enhance its penetration through the skin. Thus, the best flux values were shown by capric acid: MPS, 3:1 molar ratio, and lauric acid:MPS, 1:1 and 2:1 molar ratio, while myristic acid:MPS, 1:1 molar ratio, was the optimum under the experimental conditions used. The mechanism of the enhancing effect was examined by measuring 1H NMR spectra and the apparent partition coefficient of MPS, lauric acid, and the mixture. The apparent partition coefficient of MPS between n-octanol and water was higher for the mixture with lauric acid than for MPS alone. A 1:1 molar ratio formulation of MPS and lauric acid was selected for the in vivo permeation study. The data indicated that lauric acid increased the diffusivity of MPS in the skin by forming a complex and by affecting its partition coefficient between the skin and the delivery system.

Repartitioning effect of a mixed beta-agonist on body composition.

Subcutaneous administration of a mixed beta-agonist to young rats induced no changes in animal growth and food conversion efficiency. However, a repartitioning effect was found with increases in lean tissue and decreases in body fat. The enhancement of muscle protein deposition was attributed to a fall in protein breakdown as muscular cathepsin A activity was lower in treated rats. A reduction of muscle reduction-oxidation state is associated to those changes in protein metabolism.

Plasma levels, heart rate, and blood pressure after intravenous, oral, and aerosol administration of reproterol in man.

Three healthy volunteers received single doses of reproterol (D 1959) by means of intravenous infusion, oral and aerosol application, respectively. Administrations were separated by at least 1 week. Plasma levels were measured by means of high performance liquid chromatography. After infusion, plasma levels showed a steep decline, indicative for a rapid distribution. Even though relatively large amounts of drug were given (up to 540 micrograms), this phenomenon caused reproterol levels to drop to values near or below the limit of reliable quantitation (1 ng ml-1) within 60 to 90 min. After oral administration of one or two tablets (containing 20 mg reproterol HCl per tablet), a very short lag time could be observed, indicating fast absorption. After one tablet, plasma level maxima (of plateau-type) were 5-6 ng ml-1 and 2-3 ng ml-1 in two subjects, respectively. After two tablets, plateau level maxima around 18 ng ml-1 and 9 ng ml-1 were found, respectively. After inhalation of a metered aerosol (two puffs of 500 micrograms each) the drug appeared in plasma within minutes, albeit at very low levels, and usually remained detectable at the sub-nanogram level during the time of the experiment (2 h). Due to the very low levels and to some oscillations in the plasma concentration-time curves, a detailed pharmacokinetic assessment could not be carried out. Effects on heart rate and blood pressure were negligible. Only during the infusion of high doses (540 micrograms) was there an increase in heart rate of about 50 to 120 beats min-1. Other side-effects were also negligible.

Chronobiology and asthma. II. Body-time-dependent differences in the kinetics and effects of bronchodilator medications.

Several bronchodilator medications exhibit body-time (i.e., biological rhythm)-dependent changes in their pharmacokinetics and effects. Epinephrine (Adrenalin), metaproterenol (orciprenaline), aminophylline, and ipratropium bromide all have a better effect on the tone of the airways during the night and/or morning, when bronchial patency is low, than during the day, when it is high. The pharmacokinetics of sustained-release theophyllines (SRTs) exhibit administration-time differences. Day-night dosing-time differences in the kinetics of theophylline are especially prominent in children. Generally, in day-active asthmatic children the absorption of SRT is more rapid after a morning than an evening dosing. The administration-time effect on the kinetics of SRTs also is apparent in adult patients, but the magnitude of difference between the day versus evening administrations apparently is more moderate. Initial findings from studies of unequal (morning versus evening) BID dosing schedules--more theophylline or terbutaline before bed-time than arising--reveal a better therapeutic advantage relative to equal BID dosing schedules for those patients with predominantly nocturnal symptoms. Once-daily (OD) SRTs intended for delivery of the entire daily dose at a single time also differ quantitatively in their chronokinetics. Since asthma is mainly a nocturnal disease in many patients, it has been recommended by many that ODSRTs be taken in the evening. If taken in the morning, as is the current practice in the United States, they may not ensure therapeutic theophylline blood levels during the night when most needed. Moreover, not all ODSRTs appear suitable for once-nightly administration because of unacceptable kinetics.