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

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

Suppression of NK cell activity and of resistance to metastasis by stress: a role for adrenal catecholamines and beta-adrenoceptors.

Although acute stress has been reported to suppress natural killer cell activity (NKA) and host resistance to metastasis, it is unclear whether the sympathetic nervous system (SNS) has a role in these effects. The current study in Fischer 344 rats assessed the involvement of adrenal catecholamines and beta(1)- and beta(2)-adrenoceptors in mediating these deleterious effects of swim stress. In addition to assessing the number and activity of NK cells following swim stress, we used a tumor model based on the MADB106 mammary adenocarcinoma line: this syngeneic tumor metastasizes only to the lungs, and its lung tumor retention (LTR) and metastatic colonization are highly sensitive to NKA. The findings indicate that stress increased both LTR, assessed 24 h after inoculation, and the number of lung metastases, counted 3 weeks later. These effects were attenuated or completely abolished by the ganglionic blocker chlorisondamine (3 mg/kg i.p.), by adrenal demedullation, by a selective beta-adrenergic antagonist (nadolol, 0.4 mg/kg), and additively by a selective beta(1)- (atenolol, 1-6 mg/kg) and a selective beta(2)-antagonist (either butoxamine 4-32 mg/kg or ICI-118,551 0.3-8 mg/kg). Stress also suppressed NKA, and adrenal demedullation prevented this suppression. Administration of adrenaline (0.1-1 mg/kg) or of a beta-adrenergic agonist (metaproterenol, 0.8 mg/kg), in physiologically relevant doses, suppressed NKA in a dose-dependent manner, and increased LTR to levels characteristic of swim stress. Taken together, these findings suggest that acute stress, by releasing catecholamines from the adrenal glands and activating beta(1)- and beta(2)-adrenoceptors, suppresses NKA and consequently compromises resistance to NK-sensitive metastasis.

Use of bronchial provocation with histamine to compare the pharmacodynamics of inhaled albuterol and metaproterenol in patients with asthma.

Because measurement of effects on airway responsiveness may have advantages over the study of bronchodilatation for the evaluation of the effects of inhaled beta 2-agents, we developed a method using airway responsiveness for the independent quantitation of the relative potencies and rates of decline in effect of these drugs. This methodology was applied to the evaluation and comparison of inhaled metaproterenol and albuterol. The effects of two different doses of each drug (one and two inhalations of albuterol and two and four inhalations of metaproterenol from commercially available metered-dose inhalers) were compared with a double-blind, randomized, placebo-controlled, crossover study of 13 subjects. The effects of metaproterenol and albuterol declined at rates that were not significantly different. However, based on the effects on activity ratio at 30 minutes, each puff of metaproterenol was an estimated 0.37 times as potent as each puff of albuterol (95% confidence limits, 0.22 to 0.52). In recommended two puff doses, measurable effects of albuterol persisted longer than effects of metaproterenol. However, this appears to be because of a greater initial effectiveness of two puffs of albuterol rather than differences in the rates at which the effects of the two drugs declined with time. Airway responsiveness thus appears to be a useful tool for evaluating inhaled beta 2-agonists and designing beta 2-agonist dosing regimens.

Relative bioavailability of metaproterenol in humans utilizing a single dose, stable isotope approach.

The relative bioavailability of metaproterenol (3,5-dihydroxy-alpha-[(isopropylamino)methyl]benzyl alcohol) following a single dose (10-mg metaproterenol sulfate tablet) was studied in six normal male volunteers using coadministration of a solution of a deuterated analogue (metaproterenol-d7 sulfate). The bioavailability of the tablet formulation relative to that of the oral solution was 92 +/- 9%, with excellent power at the 5% significance level. Comparison of the coadministration of the labeled and unlabeled metaproterenol sulfate solutions in two subjects after a one-week washout demonstrated the absence of an isotope effect on either absorption or elimination. A GC-MS assay for metaproterenol was developed to measure plasma concentrations resulting from oral administration. The assay was linear over the range of 0.5-8 ng/mL, corresponding to typical plasma metaproterenol concentrations obtained after a single 10-mg oral dose. Accuracy and precision data were obtained at metaproterenol concentrations of 1.0 and 2.0 ng/mL plasma to demonstrate the applicability of the assay for bioavailability studies. Following oral administration, metaproterenol showed peak plasma concentrations of 2.2 to 13 ng/mL at 0.75 to 3.0 h, with a terminal harmonic mean half-life of 2.1 h over the plasma concentration range studied. The renal clearance of 133-158 mL/min for metaproterenol slightly exceeds the glomerular filtration rate in humans.

Effect of orally administered orciprenaline on tracheobronchial mucociliary clearance.

The relative effects of beta-2-adrenergic stimulation of mucociliary transport in the trachea and the bronchial airways was determined in 12 healthy non-smoking males using radioaerosol techniques with and without the oral administration of 20 mg of orciprenaline. Orciprenaline increased tracheal mucociliary transport rates (TMTR) from a control mean of 4.4 +/- 2.4 mm/min to 6.6 +/- 4.0 mm/min (p less than 0.01), but the percentage of particles cleared from the lung in 2 h was not significantly different in the drug study (44 +/- 14%) compared to the control study (48 +/- 19%). Furthermore, a strong positive correlation existed between TMTR and bronchial clearance that was lost after administration of orciprenaline. This study demonstrates that low doses of orally administered orciprenaline increased tracheal mucociliary transport but not bronchial clearance in healthy adults.

[Aspects of chronopharmacology in pediatrics]. / Aspects de la chronopharmacologie en pédiatrie.

Pediatric chronopharmacological findings until now have been limited to circadian changes in children 6 to 15 years old. This means that (a) data in neonates and even in infants of one year is not available and (b) other bioperiodicities with periods of several hours (ultradian rhythms) as well as several months and about one year (infradian rhythms) have not been explored in older children. Biologic time-related changes have been documented for phenytoin and theophylline with regards to pharmacokinetics and for orciprenaline and methylprednisolone with regards to effectiveness (bronchodilatation). Despite the limited number of experiments performed to date, it is already possible to admit that, (a) a chronopharmacological approach provides better precision of pharmacologic study than the conventional approach not using time-related data; (b) better therapeutics can be achieved with the help of chronopharmacological facts since an appropriate timing in administration of a medicine usually enhances its desired effects and/or reduces its undesired effects.

[Metabolism of beta-adrenergic substances. Therapeutic implications]. / Métabolisme des substances bêta-adreénergiques. Implications thérapeutiques.

The metabolism of the main beta-adrenoceptor stimulants which are not catechol derivatives involves conjugation with glucuronic or sulphuric acids in several animal species and conjugation with sulphuric acid in man. These drugs are not metabolized by MAO like isoproterenol or by COMT like the catechol derivatives: isoproterenol, trimetoquinol, hexoprenaline and rimiterol. Sulphate conjugation, in man, increases with the number of hydroxy groups. For salbutamol, pirbuterol, terbutaline and fenoterol, about 30%, 30%, 15% and 10% are respectively present in plasma as the unchanged active compound. Clenbuterol, a new specific beta 2-adrenoceptor stimulant, is a 4-amino-3,5 dichloro-benzene derivative and cannot be conjugated. It is cleared from the body mainly by the renal route (43% of the administered dose) and has eight minor metabolites, identical in several animal species and in man. Tulobuterol with no hydroxy substitute does not undergo conjugation, but is metabolized to 4-hydroxy tulobuterol. This metabolite is shown to be eight times more potent than tulobuterol. Metabolism depends greatly upon the route of administration: intravenous, subcutaneous, oral, by aerosol or instillation into the bronchial tree. Conjugation or COMT inactivation can take place in the gut wall (terbutaline), in lungs (isoproterenol, terbutaline, rimiterol) or by hepatic first-pass. These processes decrease the amount of drug reaching the blood and the receptor sites. Metabolism in the lung is important for ibuterol (terbutaline diisobutyrate), which is more lipophilic than terbutaline and spreads throughout tissues where it is hydrolyzed to active terbutaline. Biotransformations are determined by environmental or genetic factors and by the associated therapy and can change dramatically from one patient to another (interindividual variability) or for the same patient by multiple dosing (intra-individual variability). These differences in the rates of the metabolism can explain, partly, the differences observed in the response to beta-adrenoceptor stimulants by responder or non-responder patients. Decision about a therapeutic dosage regiment involves the choice of the drug, of the route of administration and of the dose. This choice is made on the basis of the dose/response relationship. In the kinetic approach, pharmacokinetic data obtained after a single dose facilitate the development of an appropriate dosage regimen.

Glucuronidation of morphine and six beta 2-sympathomimetics in isolated rat intestinal epithelial cells.

The metabolism of morphine and six beta 2-sympathomimetics (orciprenaline, terbutaline, fenoterol, salbutamol, ritodrine, and bamethan) in isolated rat intestinal epithelial cells was investigated. Only conjugates with glucuronic acid were detected. With regard to observed Vmax values the beta 2-sympathomimetics can be divided in two groups. The three resorcinols (orciprenaline, terbutaline, fenoterol) have a Vmax value comparable to that of morphine (70-230 pmol/min X mg cell protein). The phenolic beta 2-sympathomimetics (salbutamol, ritodrine, bamethan) exhibit a Vmax value comparable to the Vmax value of 1-naphthol (500-1100 pmol/min X mg cell protein). Calculation of intestinal intrinsic (metabolic) clearance and intestinal first pass extraction ratios from Vmax and Kappm values suggests that most of these drugs may undergo substantial intestinal first pass metabolism after oral administration. The glucuronidation of morphine in isolated mucosal cells could be completely inhibited by addition of 1-naphthol (50 microM), salicylamide (5 mM), or fenoterol (5 mM). Glucuronidation of fenoterol could be partially inhibited by 1-naphthol, salicylamide, and morphine. Preliminary data obtained with microsomes suggest that 1-naphthol and morphine are metabolized by different forms of the intestinal microsomal UDP-glucuronosyltransferase. Fenoterol and ritodrine are probably glucuronidated by the form, which also glucuronidates morphine. The results demonstrate that rat intestinal epithelial cells can be used to predict intestinal metabolism of morphine and other drugs at least qualitatively and that phenolic food constituents (e.g. 1-naphthol) and non-prescription drugs (e.g. salicylamide) may affect the intestinal first pass metabolism of morphine and fenoterol.

Determination of bioavailability on the basis of tachycardia after intravenous and oral administration of fenoterol, orciprenaline and salbutamol in non-anaesthetized rats.

Three beta-sympathomimetics, fenoterol, orciprenaline and salbutamol, were tested for their tachycardia effect in conscious rats sedated with brotizolam. The tachycardia after oral and intravenous administration and the time-effect curves were used for the determination of the bioavailability. In these experiments, fenoterol and orciprenaline showed better bioavailability than salbutamol. The tachycardic time effects of fenoterol and orciprenaline were of shorter duration than that of salbutamol.

The formaldehyde-donating activity of N5,N10-methylene tetrahydrofolic acid in xenobiotic biotransformation.

Reaction of reproterol, (7-(3-[2-(3,5-dihydroxyphenyl)-2-hydroxyethylamino]propyl)theophyl line (I), with formaldehyde liberated in a solution of N5,N10-methylene tetrahydrofolic acid at pH 7.3 led to the formation of 7-[3-(4,6,8-trihydroxy-1,2,3,4-tetrahydroisoquinolinyl-2)propyl]th eophylline (II), but no such reaction was observed in a solution of N5-methyl tetrahydrofolic acid. Compound II was also formed by incubation of I with rat-liver subcellular fractions. The highest formaldehyde-donating activity was found in the soluble fraction but some activity was also observed with washed mitochondria and microsomes. Dimedone was transformed to methylene bisdimedone by incubation with the soluble fraction. The amount of formaldehyde liberated in the soluble fraction during a four hour incubation at 22 degrees C was 1-3.6 mumol/g of liver. Metaproterenol and terbutaline, structurally related to I, reacted with formaldehyde to yield the corresponding tetrahydro-isoquinolines. The rate of this chemical reaction for the three drugs correlated with the amount of the tetrahydroquinolines formed by incubation with the soluble fraction and in orally dosed rats.

Isolation and characterization of metaproterenol-3-O-sulfate: a conjugate of metaproterenol in human urine.

Metaproterenol (1-(3,5-dihydroxyphenyl)-2-isopropylaminoethanol) is primarily converted in humans to metaproterenol-3-O-sulfate following oral administration. Ion exchange column chromatography with a gradient of ammonium acetate buffer permitted the isolation of the ammonium salt of metaproterenol-3-O-sulfate from human urine. Treatment of aliquots of the column eluate with purified sulfatase and subsequent HPLC/fluorescence analysis confirmed the presence of metaproterenol. Comparison of the column eluate with a metaproterenol standard by 250-MHz proton-NMR revealed a pattern consistent with monosubstitution of the resorcinol ring. Negative and positive ion fast atom bombardment/mass spectrometry showed the metabolite to have a (M-H)- m/z of 290 and a (M + H)+ m/z ion of 292. These three methods support the structural assignment of metaproterenol-3-O-sulfate. Enzymatic hydrolysis of urine specimens from 29 different subjects with purified beta-glucuronidase as well as beta-glucuronidase-sulfatase mixtures yielded no significant increase in metaproterenol beyond purified sulfatase-treated urine, thus ruling out the presence of a glucuronide of metaproterenol. Approximately 40% of an oral 20-mg dose, given as either a tablet or a solution, was recovered in the urine as metaproterenol-3-O-sulfate. Approximately 5% of the dose was recovered in the unconjugated form. The majority of the dose was excreted over the first 12 hr with a biological half-life of 5-6 hr followed by a slower excretion phase with a half-life of 20 hr.

Dissociation constants of isoprenaline and orciprenaline and their relative efficacies on guinea-pig isolated atria determined by use of an irreversible beta-adrenoceptor antagonist.

1. Dissociation constant (KA) of isoprenaline and orciprenaline were determined for the positive inotropic and chronotropic responses of guinea-pig isolated atria. Cumulative dose-response curves to the agonists were constructed before and after incubation with and washout of the irreversible beta-adrenoreceptor antagonist, Ro 03-7894 1-(5-chloracetylaminobenzfuran-2-yl)-2-isopropylaminoethanol). 2. After 20 min washout, the curves were displaced to the right with depression of the maxima. After 3 h washout, there was only depression of the maxima. 3. Dissociation constants were determined by plotting reciprocals of molar concentrations before Ro 03-7894 (1/A) against reciprocals of the equiactive concentrations after Ro 03-7894 (1/A'). KA = (slope - 1)/ intercept. 4. Isoprenaline had a greater affinity (KA) than orciprenaline on both rate and tension. The affinity for rate and tension was identical for both agonists, indicating that the beta-adrenoceptors were identical. 5. Isoprenaline and orciprenaline produced identical rate response maxima when compared in the same preparation but the orciprenaline tension maximum was only 92.0 +/- 0.3% that of isoprenaline. 6. These dose-response curves were replotted as response against -log RA/Rt (fraction of receptors occupied) for each agonist concentration, calculated from the equation RA/Rt = (A)/(KA + (A)). The antilogarithm of the distance along the -log RA/Rt axis gave the efficacy of orciprenaline relative to isoprenaline. It had a greater efficacy (2.24) for rate but a lower efficacy for tension responses (0.5).

Biotransformation of reproterol in the intestinal tract of the rat.

The major metabolite of reproterol (Broncho-spasmin) in rat feces is 2-[3-theophyllinyl(7)-propyl]-4,6,8-trihydroxy-1,2,3,4-tetrahydroisoquinoline. It was also found in the bile of orally or intravenously dosed rats in the form of glucuronides. The biotransformation of an oral dose of reproterol to this metabolite occurred mostly in the cecum-colon section of the intestinal tract. Experiments in antibiotic treated rats showed no significant effect of the treatment on the extent of metabolite formation. This metabolite was also formed by incubation of reproterol with cecum-colon homogenates under aerobic as well as anaerobic conditions. The pharmacological action after an oral dose must be attributed to reproterol absorbed as intact form, since the metabolite is inactive.

[Plasma levels, renal excretion and metabolism of orciprenaline after administration in sustained-release form (author's transl)]. / Plasmaspiegel, renale Ausscheidung und Metabolismus von Orciprenalin nach Verabreichung als Depotpräparat.

A newly developed sustained-release form of orciprenaline-sulfate (Alupent) was tested in 13 patients. Determination of 3H-radioactivity in blood, urine and faeces was used to elucidate its pharmacokinetic properties. Maximum plasma levels of radioactivity were obtained between 8 and 12 h after administration. 10.7 +/- 2.5% of the administered radioactivity were excreted in urine over a period of 72 h. Orciprenaline was mainly excreted as the sulfate-conjugate. Approximately five percent of the radioactivity were excreted as 4,6,8-trihydroxy-N-isopropyl-tetrahydroisoquinoline--the condensation product of formaldehyde and orciprenaline.

[Studies on the biotransformation of reproterol. Structure determination of the main metabolite (author's transl)]. / Untersuchungen zur Biotransformation von Reproterol. Strukturaufklärung des Hauptmetaboliten.

Biotransformation of 7-(3-[2-(3,5-dihydroxyphenyl)-2-hydroxy-ethylamino]-propyl)-theophylline (reproterol, Bronchospasmin), a beta 2-adrenergic drug recently introduced into therapeutic use, leads to the same main metabolite in animals and in man. By mass-spectroscopy and by synthesis its structure was shown to be tetrahydroisoquinoline derivative which is produced from reproterol by uptake of an additional carbon atom concomitant with cyclization.

Metabolism and pharmacokinetics of orciprenaline in various animal species and man.

The physiological disposition of tritiated orciprenaline, the 3,5-dihydroxy-isomer of isoprenaline, has been studied in rat, rabbit, cat and man. In the lungs, liver and kidneys of the rat higher 3H-concentrations were found than in the plasma after i.v. injection. A higher percentage of unchanged drug was located in the heart, lungs and skeletal muscle only. In the plasma of rabbit and cat as well as man the decline of 3H-activity was biphasic after i.v. administration with a half-life of 15 hr in the rabbit 6 hr in cat and man for the slower phase. The pharmacokinetic analysis of the post-infusion data in man resulted in an open two-compartment model with a high volume of distribution (ca. 700 liters). The total clearance (1400 ml/min) was rather high. The renal clearance contributed to 10 per cent only. The amount of drug absorbed after oral administration amounted to 44%, the reduced biovailability of 10 per cent indicated a fist-pass effect. The metabolism lead only to conjugates with glucuronic acid, most probably at one of the phenolic hydroxyl groups in rat, rabbit and man. The cat did not metabolize orciprenaline to an appreciable amount.