Pharmacogenetic characterization of indacaterol,a novel β2-adrenoceptor agonist

Background and purpose:

Indacaterol is a novel β₂-adrenoceptor agonist in development for the treatment of chronic obstructive pulmonary disease. The aim of this study was to investigate the comparative pharmacology of indacaterol in recombinant cells expressing the common polymorphic variants of the human β₂-adrenoceptor and in human primary airway smooth muscle (ASM) cells.

Experimental approach:

Chinese hamster ovarian-K1 cell lines expressing high and low levels of the common human β₂-adrenoceptor variants were generated [Gly16-Glu27-Val34-Thr164(GEVT), RQVT, GQVT] and also the rare GQVI variant. Human primary ASM cells were isolated from explants of trachealis muscle. Adenosine-3′,5′-cyclic-monophosphate production was used as an outcome measure.

Key results:

In both the low- and high-expression recombinant GEVT ‘wild type’ cell lines indacaterol is a high-efficacy agonist. Salmeterol and formoterol were identified as low- and high-efficacy agonists, respectively, and showed similar potencies to indacaterol irrespective of the β₂-adrenoceptor genotype. The I164 variant cell line was associated with a reduced capacity to generate adenosine-3′,5′-cyclic-monophosphate in response to β₂-adrenoceptor agonist. In the human primary ASM cells indacaterol gave a maximal response intermediate between that of salmeterol and formoterol.

Conclusions and implications:

These data demonstrate that indacaterol is a high-efficacy agonist in recombinant cell systems but acts with lower efficacy in human primary ASM cells. No marked genotype-dependent effects were observed for common variants; however, changes in I164 receptor activity were identified, which were dependent on the level of expression of β₂-adrenoceptors.     

Formoterol and salmeterol induce a similar degree of β2-adrenoceptor tolerance in human small airways but via different mechanisms

BACKGROUND AND PURPOSE

Steroids prevent and reverse salbutamol-induced β₂-adrenoceptor tolerance in human small airways. This study examines the effects of the long-acting β₂ agonists (LABAs) formoterol and salmeterol, and the ability of budesonide to prevent desensitization.

EXPERIMENTAL APPROACH

Long-acting β₂ agonists in the presence and absence of budesonide were incubated with human precision-cut lung slices containing small airways. Tolerance was deduced from measurements of reduced bronchodilator responses to isoprenaline and correlated with β₂-adrenoceptor trafficking using a virally transduced, fluorescent-tagged receptor. The ability of the LABAs to protect airways against muscarinic-induced contraction was also assessed.

KEY RESULTS

Following a 12 h incubation, both formoterol and salmeterol attenuated isoprenaline-induced bronchodilatation to a similar degree and these effects were not reversible by washing. Pre-incubation with budesonide prevented the desensitization induced by formoterol, but not that induced by salmeterol. Formoterol also protected the airways from carbachol-induced bronchoconstriction to a greater extent than salmeterol. In the epithelial cells of small airways, incubation with formoterol promoted receptor internalization but this did not appear to occur following incubation with salmeterol. Budesonide inhibited the formoterol-induced reduction in plasma membrane β₂-adrenoceptor fluorescence.

CONCLUSIONS AND IMPLICATIONS

Although both formoterol and salmeterol attenuate isoprenaline-induced bronchodilatation, they appear to induce β₂-adrenoceptor tolerance via different mechanisms; formoterol, but not salmeterol, enhances receptor internalization. Budesonide protection against β₂-adrenoceptor tolerance was correlated with the retention of receptor fluorescence on the plasma membrane, thereby suggesting a mechanism by which steroids alter β₂-adrenoceptor function.     

Portal hypertension and liver cirrhosis in rats: effect of the β3-adrenoceptor agonist SR58611A

BACKGROUND AND PURPOSE

β₃-Adrenoceptors participate in the regulation of vascular tone in physiological and pathological conditions. We aimed to assess the effect of pharmacological modulation of β₃-adrenoceptors on portal pressure (PP) and systemic haemodynamics and their expression in the liver and mesenteric vessels of cirrhotic rats.

EXPERIMENTAL APPROACH

PP, central venous pressure (CVP) and systemic haemodynamics were invasively assessed in control and CCl₄-treated cirrhotic rats before and during infusion of the selective β₃-adrenoceptor agonist, SR58611A. Tissue samples were also collected from liver, heart, portal vein and mesenteric artery for immunohistochemistry and molecular biology analysis. The effect of SR58611A on isolated portal vein was assessed.

KEY RESULTS

At baseline, cirrhotic rats showed portal hypertension, reduced CVP and hyperdynamic circulation. SR58611A induced a significant, dose-dependent decrease in PP in cirrhotic rats, but not in controls. Although both groups manifested a dose-dependent reduction in mean arterial pressure, this effect was associated with decreased cardiac index (CI) and unchanged indicized peripheral vascular resistance (PVRI) in cirrhotic rats and increased CI and decreased PVRI in control animals. Pretreatment with the selective β₃-adrenoceptor antagonist SR59230 prevented all SR58611A-induced changes in cirrhotic rats. SR58611A concentration-dependently relaxed portal vein in cirrhotic rats to a significantly greater extent than in healthy rats; pretreatment with SR59230A completely prevented SR58611A-induced cirrhotic portal vein relaxation. Finally, β₃-adrenoceptors were identified in the liver, heart and portal vein of cirrhotic and control animals; their expression was increased in cirrhotic rats.

CONCLUSIONS AND IMPLICATIONS

β₃-Adrenoceptors are altered in portal hypertension of experimental cirrhosis and may represent a novel therapeutic target.     

Pharmacological profile of the selective β3-adrenoceptor agonist mirabegron in cynomolgus monkeys

Mirabegron is a novel β₃-adrenoceptor agonist developed for the treatment of overactive bladder. To clarify the relationship between the pharmacological effects of mirabegron in monkeys and the clinical efficacy in patients with overactive bladder, the effect of mirabegron on bladder function was evaluated using cynomolgus monkeys. Quantitative PCR revealed that mRNA expression of β₃-adrenoceptors was most abundant (98 %) among β-adrenoceptor subtypes in the bladder of cynomolgus monkeys. Mirabegron, which showed selective and potent agonistic activity on monkey β₃-adrenoceptors expressed in Chinese hamster ovary cells with EC₅₀ value of 32 nmol/L and intrinsic activity of 0.8, induced concentration-dependent relaxation of bladder smooth muscle strips isolated from cynomolgus monkeys with EC₅₀ values of 120 nmol/L in 20 mmol/L KCl stimulation and 43 nmol/L under 9.81 mN resting tension. In conscious cynomolgus monkeys, mirabegron decreased micturition frequency at oral doses of 1 and 3 mg/kg and increased mean volume voided per micturition at an oral dose of 3 mg/kg. Plasma concentration at which bladder function improved in the cynomolgus monkeys was similar to that at the clinically effective dose in patients with overactive bladder. These data suggest that the relaxant function in monkey bladder is mainly mediated by β₃-adrenoceptors similar to that in the human bladder and mirabegron showed efficacy on the bladder functions of the same parameters in clinical evaluation endpoints.     

The pharmacokinetics of the β2-adrenoceptor agonist,tulobuterol, given transdermally and by inhalation

Summary We have studied the pharmacokinetics of tulobuterol given transdermally or by aerosol inhalation in healthy male volunteers.Tulobuterol was rapidly absorbed after inhalation, with a t_max of 0.8–1.5 h. The C_max and the AUC increased linearly with dose.Tulobuterol was well absorbed after transdermal administration, with an absorption lag-time of about 4 h. The C_max and AUC increased linearly with dose and the t_max was about 9–12 h. The mean percentage of drug absorbed during the application of a patch for 24 h was 82–90% after a single dose and 82–85% during repeated dosing.The mean urinary recoveries as unchanged drug after a single inhalation and patch application were 3–4% and 5–6% respectively.Tulobuterol did not accumulate during repeated inhalation or transdermal application. It was well tolerated, except for an increase in heart rate of 10–20 beats · min^–1 after five repeated applications of a 4 mg patch.     

The long-acting β2-adrenoceptor agonist olodaterol attenuates pulmonary inflammation

Background and Purpose

β₂-adrenoceptor agonists are widely used in the management of obstructive airway diseases. Besides their bronchodilatory effect, several studies suggest inhibitory effects on various aspects of inflammation. The aim of our study was to determine the efficacy of the long-acting β₂-adrenoceptor agonist olodaterol to inhibit pulmonary inflammation and to elucidate mechanism(s) underlying its anti-inflammatory actions.

Experimental Approach

Olodaterol was tested in murine and guinea pig models of cigarette smoke- and LPS-induced lung inflammation. Furthermore, effects of olodaterol on the LPS-induced pro-inflammatory mediator release from human parenchymal explants, CD11b adhesion molecule expression on human granulocytes TNF-α release from human whole blood and on the IL-8-induced migration of human peripheral blood neutrophils were investigated.

Key Results

Olodaterol dose-dependently attenuated cell influx and pro-inflammatory mediator release in murine and guinea pig models of pulmonary inflammation. These anti-inflammatory effects were observed at doses relevant to their bronchodilatory efficacy. Mechanistically, olodaterol attenuated pro-inflammatory mediator release from human parenchymal explants and whole blood and reduced expression of CD11b adhesion molecules on granulocytes, but without direct effects on IL-8-induced neutrophil transwell migration.

Conclusions and Implications

This is the first evidence for the anti-inflammatory efficacy of a β₂-adrenoceptor agonist in models of lung inflammation induced by cigarette smoke. The long-acting β₂-adrenoceptor agonist olodaterol attenuated pulmonary inflammation through mechanisms that are separate from direct inhibition of bronchoconstriction. Furthermore, the in vivo data suggest that the anti-inflammatory properties of olodaterol are maintained after repeated dosing for 4 days.Tables of Links

Effects of chronic treatment with the new ultra-long-acting β2-adrenoceptor agonist indacaterol alone or in combination with the β1-adrenoceptor blocker metoprolol on cardiac remodelling

Background and Purpose

The ability of a chronic treatment with indacaterol, a new ultra-long-acting β₂-adrenoceptor agonist, to reverse cardiac remodelling and its effects in combination with metoprolol, a selective β₁-adrenoceptor antagonist, were investigated on myocardial infarction in a rat model of heart failure (HF).

Experimental Approach

We investigated the effects of indacaterol and metoprolol, administered alone or in combination, on myocardial histology, β-adrenoceptor-mediated pathways, markers of remodelling and haemodynamic parameters in a rat model of HF. Five groups of rats were assessed: sham-operated rats; HF rats; HF + indacaterol 0.3 mg·kg⁻¹·day⁻¹; HF + metoprolol 100 mg·kg⁻¹·day⁻¹; HF + metoprolol + indacaterol. All pharmacological treatments continued for 15 weeks.

Key Results

Treatment with either indacaterol or metoprolol significantly reduced the infarct size in HF rats. However, the combination of indacaterol and metoprolol reduced the infarct size even further, reduced both BP and heart rate, reversed the decrease in ejection fraction, normalized left ventricular systolic and diastolic internal diameters, normalized the decreased β₁ adrenoceptor mRNA expression as well as cardiac cAMP levels and reduced cardiac GPCR kinase 2 expression, compared with the untreated HF group.

Conclusion and Implications

The results of our study demonstrated an additive interaction between indacaterol and metoprolol in normalizing and reversing cardiac remodelling in our experimental model of HF. The translation of these findings to clinical practice might be of interest, as this combination of drugs could be safer and more effective in patients suffering from HF and COPD.Tables of Links

On the mechanism of the persistent action of salmeterol: what is the current position?

The mechanism of the long duration of action of salmeterol at β₂-adrenoceptors has long been a matter of debate, and is still unresolved. Szczuka and colleagues have both summarized the position to date and suggested a new mechanistic contender, receptor rebinding. Despite this, they still do not come to any clear conclusion. Much of the literature data that they have drawn upon appears contradictory, and mathematical models are inevitably flawed by the questionable validity of key values applied to them. Although the issue will undoubtedly eventually be resolved, it will probably require investigators to apply carefully designed studies on simple experimental systems such as isolated membranes or cultured cells. Only then should studies be extended to more complex systems such as isolated preparations of airways smooth muscle, where tissue bulk inevitably presents a complicating factor, particularly where relatively lipophilic compounds are concerned.     

Protective effects of the β3-adrenoceptor agonist CL316243 against N-methyl-D-aspartate-induced retinal neurotoxicity

We have previously reported that β₃-adrenoceptor agonists dilate retinal blood vessels, but their effects on retinal neurons have been unclear. In this study, we examined the action of the β₃-adrenoceptor agonist CL316243 against retinal damage induced by intravitreal injection of N-methyl-D-aspartate (NMDA) in rats. CL316243 was injected into the vitreous cavity before, with, or after intravitreal NMDA injection. Seven days after NMDA injection, cell loss in the ganglion cell layer (GCL) and thinning of the inner plexiform layer were observed. The reduction in the number of cells in the GCL was diminished by injection of CL316243 at 15, 30, 60, or 120?min after NMDA injection, whereas no significant protective effect was observed when CL316243 was administered 240?min after NMDA injection. Neither preinjection of CL316243 30?min before NMDA nor simultaneous injection of CL316243 with NMDA exerted any protective effect. The β₃-adrenoceptor antagonist L748337 almost completely abolished the protection conferred by CL316243 injection 120?min after NMDA injection. The number of parvalbumin-positive amacrine cells was decreased in eyes examined 1?day after NMDA treatment, but this was prevented by CL316243 injection at 120?min after NMDA injection. These results suggest that CL316243 exerts protective effects against NMDA-induced damage by stimulation of β₃-adrenoceptors. β₃-adrenoceptor agonists may be effective candidates for the treatment of retinal diseases associated with glutamate-induced excitotoxicity, including glaucoma and diabetic retinopathy.     

The pharmacokinetics of theβ 2-adrenoceptor agonist fenoterol in healthy women

Summary We have studied the pharmacokinetics of fenoterol in healthy women during and after a 3 h intravenous infusion of different doses within the therapeutic range for tocolysis (0.5 g·min^–1, 1.0 g·min^–1, and 2.0 g·min^–1). A specific and sensitive radioimmuno-assay was used for the determination of fenoterol. For compartmental analysis the plasma concentration time data were fitted with the TOPFIT program, assuming two exponentials.The total clearance of fenoterol increased with dose (1299 ml·min^–1 at 0.5 g·min^–1, 1483 ml·min^–1 at 1.0 g·min^–1, and 1924 ml·min^–1 at 2.0 g·min^–1), as did the apparent volume of distribution (from 491 at the lowest to 851 at the highest dose).In contrast, the apparent half-lives were not dose-dependent, with t_{1/2· 1} 4.8 min and t_{1/2· 2} 52 min.This paper is dedicated to Prof. Dr. Ellen Weber, Heidelberg, FRG     

Lack of evidence that nebivolol is a β₃-adrenoceptor agonist

Nebivolol is a selective β?-adrenoceptor antagonist which, in addition, displays endothelium-dependent vasodilating properties in humans and other species. β?-adrenoceptors have been proposed to be a molecular target of nebivolol-induced vasodilatation. Therefore, we have investigated possible β?-adrenoceptor agonism by nebivolol for relaxation of the human and rat urinary bladder (prototypical β?-adrenoceptor-mediated responses) as well as for cAMP accumulation in Chinese hamster ovary cells stably transfected with the human β-adrenoceptor subtypes. Nebivolol concentration-dependently relaxed both human and rat isolated urinary bladder strips but with low potency, similar to that reported for vasodilatation. However, nebivolol-induced bladder relaxation in either species was not inhibited by the β?-adrenoceptor antagonist SR 59,230A (10μM), although this compound inhibited the isoprenaline-induced relaxation with the expected potency. In radioligand binding studies nebivolol had lower affinity for human β?-adrenoceptors than the other two β-adrenoceptor subtypes, but this low affinity was in line with its potency to relax the bladder or isolated blood vessels. In functional studies nebivolol even in high concentrations did not stimulate cAMP formation via any of the three cloned human β-adrenoceptors or in rat bladder smooth muscle cells. Taken together these data demonstrate that nebivolol can relax not only vascular but also urinary bladder smooth muscle. However, they do not support the hypothesis that nebivolol is an agonist at cloned human β?-adrenoceptors or in rat or human urinary bladder.     

Comparison of three radioligands for the labelling of human β-adrenoceptor subtypes

We have compared the ability of three radioligands, [¹²⁵I]-cyanopindolol, [³H]-CGP 12,177 and [³H]-dihydroalprenolol, to label the three human β-adrenoceptor subtypes. Saturation and competition binding experiments were performed using membrane preparations from Chinese hamster ovary cells stably transfected with the three subtypes. While [³H]-CGP 12,177 had very similar affinity for β₁- and β₂-adrenoceptors (about 40 pM), [¹²⁵I]-cyanopindolol and [³H]-dihydroalprenolol had 4- to 6-fold higher affinity for β₂- as compared to β₁-adrenoceptors (10 vs 45 and 187 vs 1,021 pM, respectively). The affinity of [¹²⁵I]-cyanopindolol at β₃-adrenoceptors was considerably lower (440 pM) than at the other two subtypes. The β₃-adrenoceptor affinity of [³H]-CGP 12,177 and [³H]-dihydroalprenolol was so low that it could not be estimated within the tested range of radioligand concentrations (up to 4,000 pM and 30,000 pM for [³H]-CGP 12,177 and [³H]-dihydroalprenolol, respectively). We conclude that all three radioligands are ill-suited to label β₃-adrenoceptors, particularly in preparations co-expressing multiple subtypes. In the absence of alternatives, [¹²⁵I]-cyanopindolol appears the least unsuitable to label β₃-adrenoceptors. There is a need for high-affinity radioligands which are either selective for β₃-adrenoceptors or reasonably non-selective among all three β-adrenoceptor subtypes.     

Comparative trough effects of formoterol and salmeterol on lymphocyte β2-adrenoceptor – regulation and bronchodilatation

Objectives: The primary aim of the present study was to evaluate comparative trough effects of formoterol and salmeterol on β₂-adrenoceptor regulation and bronchodilator response after regular twice-daily treatment, with a secondary aim to evaluate any possible association with β₂-adrenoceptor polymorphism. Methods: Sixteen asthmatic subjects, with mean (SD) age 33(9) years, all taking inhaled corticosteroids and with a forced expiratory volume in 1 s (FEV₁) of 81(12)% predicted were recruited to take part in a randomised single-blind, three-way cross-over study. The subjects received three treatments each for 1 week, with 1-week washout periods in between: (1) formoterol dry powder, 12 μg twice daily, (2) salmeterol dry powder, 50 μg twice daily, or (3) placebo, twice daily. Spirometry and lymphocyte β₂-adrenoceptor parameters were measured before the first dose and 12 h after the last dose of each treatment, as well as domiciliary peak flow during each treatment. Results: There were no differences in β₂-adrenoceptor density (B_max) between the three treatments prior to the first dose; whereas, after the last dose, B_max was lower with both active treatments than with placebo, but was significant for salmeterol only – a 1.2-fold geometric mean fold difference (95% CI 1- to 1.4-fold), P = 0.04. Compared with placebo, there were n = 9 of 16 subjects with salmeterol and n = 6 of 16 with formoterol who had a greater than 15% fall in B_max. Post-hoc trend analysis of polymorphism showed that the propensity for downregulation appeared to be related to the occurrence of an allelic substitution of glycine at codon 16 – 8 of 13 for salmeterol versus 5 of 13 for formoterol with a greater than 15% fall compared with placebo. There were no significant differences between salmeterol and formoterol in terms of mean or individual values for downregulation. There was evidence of persistent bronchodilator activity with both active treatments compared with placebo; this was significant for forced expiratory flow rate between 25% and 75% of vital capacity (FEF_25–75) – the mean difference versus salmeterol was 0.39 l/s (95% CI 0.06–0.70), P = 0.02, and versus formoterol was 0.35 l/s (95% CI 0.16–0.53), P = 0.001. These effects were mirrored by significant improvements in morning peak flow rate compared with placebo – mean difference versus salmeterol was 24 l/min (95% CI 7–42), P = 0.01, and versus formoterol was 36 l/min (95% CI 25–48), P < 0.0001. Conclusion: There were no differences between regular treatment with formoterol and salmeterol in their effects on lymphocyte β₂-adrenoceptor regulation at the end of a 12-h dosing interval, with both drugs exhibiting a residual degree of bronchodilator activity at the same time point. Further studies to evaluate receptor regulation and bronchodilator response are required in susceptible patients who have the homozygous glycine-16 polymorphism. Received: 15 January 1999 / Accepted in revised form: 19 April 1999     

Role of β2-adrenoceptor blockade and circulating adrenaline level for the pressor responses to β-adrenoceptor blocking drugs in rats

Summary The present experiments were designed to elucidate what mechanism(s) would be responsible for -adrenoceptor blocking drugs (-blockers)-induced pressor responses in rats. In urethane-anaesthetized rats, 6 -blockers at i.v. doses ranging from 0.3 to 300 g/kg evoked the pressor response in a dose-dependent manner. The relative potency in causing the pressor action was correlated not to their ₁-blocking activities (r=0.374, P>0.05) but to their ₂-blocking ones (r=0.856, P<0.05). In pithed or adrenalectomized rats with low levels of plasma catecholamines, however, propranolol failed to exert its sustained pressor action. Propranolol (300 g/kg i.v.) distinctly potentiated the pressor responses not to noradrenaline but to adrenaline and to electrical stimulation of the sympathetic outflow (E.S.) in pithed rats. On the contrary, there was not any potentiation of pressor response to E.S. in pithed, adrenalectomized rats treated with propranolol (300 g/kg i.v.). In rats treated with phenoxybenzamine (5 mg/kg i.v.), adrenaline was shown to have much more potent vasodilating action resulting from ₂-stimulation than noradrenaline, the dose difference for causing the diastolic blood pressure decrease by a 25 mm Hg being almost 80 times. In pithed rats, infusion of adrenaline at the rate of 0.02 g/min caused a significant increase in plasma adrenaline level from 0.02±0.01 to 0.45±0.048 ng/ml, being close to basal level obtained in urethane-anaesthetized rats. Under this situation, propranolol (1–100 g/kg i.v.) showed a distinct pressor response in a dose-dependent fashion as observed in adrenal intact rats anaesthetized with urethane. These results strongly suggest that the pressor responses to -blockers are largely due to both their inhibitory actions on the vasodilatory ₂-adrenoceptors and to their potentiating actions on the vascular response to circulating adrenaline.     

Properties of agonist binding at theβ-adrenoceptor of the rat reticulocyte

Summary To study the fundamental differences between agonist and antagonist interaction with the -adrenoceptor of the rat reticulocyte the radiolabeled agonist³H hydroxybenzylisoprenaline (³H HBI) and the radiolabeled antagonist³H dihydroalprenolol (³H DHA) were used.Equilibrium binding experiments with³H HBI revealed all characteristics expected to a -adrenoceptor site, i. e. high affinity binding (K _{D high}=7.4±0.9×10^–9 M), saturability (B _{max high}=230±24 fmoles/mg protein), and stereoselectivity. The rank order of potency for competing agonists was isoprenaline > adrenaline > noradrenaline > dopamine.³H HBI high affinity binding sites amounted to about 25% of -adrenoceptor sites detectable with³H DHA.In competition experiments with³H HBI and (-)isoprenaline[(-)Ipn]aK _{D high}-value for (-)Ipn of 3.1±0.6×10^–8M was obtained corresponding to theK _{D high}-value of (-)Ipn obtained from competition experiments using³H DHA. For (-)propranololK _D-values of 0.9±0.5×10^–8 M and 1.0 ±1.0×10^–8 M were measured using³H HBI and³H DHA respectively.Agonist affinity derived from competition experiments with (-)Ipn versus³H DHA was not affected by temperature changes.Guanylyl-imidodiphosphate [Gpp(NH)p] decreased concentration dependently the number of high affinity binding sites of³H HBI not affecting the respectiveK _D-value. Similar effects were observed after omission of Mg²⁺ from the binding assay or inclusion of Na⁺ in the Mg²⁺-free incubation mixture.The association reaction of³H HBI at the -adrenoceptor revealed two different velocities. The slower phase of the association reaction which represents high affinity binding (80% of equilibrium binding) is not observed in the presence of Gpp(NH)p.A biphasic dissociation of³H HBI binding was induced by 10^–4 M (±)propranolol: 25% dissociated with at _1/2 of 1.3 min whereas the high affinity binding was reversed with at _1/2 of 150 min. This slowly reversible binding of³H HBI however was rapidly reversed by Gpp(NH)p (t _1/2<1 min).It is concluded that the agonist ligand³H HBI permits a direct qualitative and quantitative characterization of the agonist induced high affinity state of the -adrenoceptor. In particular, the kinetic studies strongly support a two step binding model for the agonist--adrenoceptor interaction.This work was supported by a grant from the Deutsche ForschungsgemeinschaftParts of this work were presented at the Spring Meetings of the German Pharmacological Society (Wiemer et al. 1978, 1981 b)Herrn Professor Dr. med. Hans Herken, Pharmakologisches Institut der Freien Universität Berlin, zum 70. Geburtstag gewidmet.     

The duration of action of non-β2-adrenoceptor mediated responses to salmeterol

1. To investigate further the mechanism of the long duration of action of the selective β₂-adrenoceptor agonist, salmeterol, we have determined the duration of action of some responses to salmeterol which are not mediated throughβ₂-adrenoceptors.
2. In the presence of propranolol (1 μM), salmeterol (1–30 μM) caused concentration-related relaxation of superfused, pre-contracted strips of guinea-pig gastric fundus. On washing the tissues, these relaxant responses were rapidly lost, the time to 50% recovery being approximately 30 min.
3. In human neutrophils, salmeterol (1–100 μM) caused concentration-related inhibition of FMLP-induced O₂⁻ release. Propranolol (1 μM) had little or no effect on the inhibitory activity of salmeterol. Washing the cells twice over a 40 min period caused a marked reduction of the inhibitory activity of salmeterol.
4. In guinea-pig superfused trachea, in the absence of propranolol, infusions of (RS)-salmeterol (10–30 nM) and the less potent (S)-enantiomer of salmeterol (300–3000 nM) inhibited electrically-induced contractile responses. When the infusion was stopped, there was no recovery from the inhibitory responses within 200 min. In the presence of propranolol (1 μM), infusions of (RS)-salmeterol (10 μM) and (S)-salmeterol (10–100 μM) also inhibited the contractile responses, but, in contrast, on stopping the infusions differences were observed in recovery times. Thus no appreciable recovery was observed from the responses to (RS)-salmeterol, whereas a rapid loss of inhibition was observed on stopping the infusion of (S)-salmeterol, the time to 50% recovery being 30–35 min.
5. These relatively short-lasting effects of salmeterol which are not mediated through β₂-adrenoceptors, contrast with the persistence of the responses which are mediated through β₂-adrenoceptors seen in a variety of tissues, but are similar to the rate of dissociation of salmeterol observed from artificial membranes. These observations suggest that the sustained agonist activity of salmeterol is peculiar to responses mediated by β₂-adrenoceptors.

     

β-Adrenoceptor regulation and functional responses in the guinea-pig following chronic administration of the long-acting β2-adrenoceptor agonist formoterol

Formoterol is a long acting ₂-adrenoceptor agonist designed for the alleviation of the symptoms of asthma. This study examined the effects of 14 day administration of formoterol (200 g/kg/day i.p.) on ₁- and ₂-adrenoceptors in guinea-pig cardiac and lung tissue. Quantitative autoradiography was used to measure changes in receptor density and organ bath studies determined alterations in functional response.Formoterol treatment produced marked reductions of between 43% and 77% in ₂-adrenoceptor density in all regions of the heart (atrioventricular node, bundle of His, right and left bundle branches, interventricular and interatrial septa, right and left atria, ventricles and apex) and lung (bronchial and vascular smooth muscle and parenchyma) (P < 0.01, n = 6). ₁-Adrenoceptor density remained unchanged in all cardiac and lung regions. In functional studies (–)-isoprenaline was 4 fold less potent at causing relaxation of carbachol (1 M) precontracted tracheal smooth muscle (pD₂: control 8.49 ± 0.03, formoterol 7.91 ± 0.10, P < 0.001, n = 4), but formoterol treatment did not change the ability of (–)-isoprenaline to elicit a maximum response. The pK_B values for ICI 118,551, 7.33 ± 0.08 in the control and 7.20 ± 0.01 in formoterol treated animals, were between those expected for ₁- and ₂-adrenoceptors suggesting involvement of both subtypes in the response. In spontaneously beating right atria and electrically paced left atria, tissues in which responses are largely mediated by ₁-adrenoceptors, there was no significant change in responses to (–)isoprenaline (right atria pD₂: control 8.45 ± 0.02; formoterol 8.42 ± 0.11; P = 0.77, n = 4) (left atria pD₂: control 8.25 ± 0.03; formoterol 8.47 ± 0.08; P = 0.09, n = 4). In the presence of CGP 20712A (100 nM) the pK_B values did not change with formoterol treatment (left atria: control 9.59 ± 0.12, formoterol 9.66 ± 0.12; P = 0.70, n = 4) (right atria: control 8.93 ± 0.11, formoterol 9.11 ± 0.07; P = 0.25, n = 4).The doses and route of administration of formoterol used in this study differs from those used clinically. However, this study demonstrates that chrome formoterol administration produces selective down-regulation of ₂-adrenoceptors in the lung and heart. The changes in the lung are accompanied by a shift to the right in the concentration-response curve to -agonist stimulation with no change in the maximum response.     

Cardiovascular effects of chronic treatment with a β2-adrenoceptor agonist relieving neuropathic pain in mice

Neuropathic pain is often a chronic condition, disabling and difficult to treat. Using a murine model of neuropathic pain induced by placing a polyethylene cuff around the main branch of the sciatic nerve, we?have shown that chronic treatment with β-AR agonists is effective against neuropathic allodynia. β-mimetics are widely used against asthma and chronic obstructive pulmonary disease and may offer an interesting option for neuropathic pain management. The most prominent adverse effects of chronic treatment with β-mimetics are cardiovascular. In this study, we compared the action of low doses of the selective β(2)-AR agonist terbutaline and of a high dose of the mixed β(1)/β(2)-AR agonist isoproterenol on cardiovascular parameters in a neuropathic pain context. Isoproterenol was used as a positive control for some heart-related changes. Cardiac functions were studied by echocardiography, hemodynamic measurements, histological analysis of fibrosis and cardiac hypertrophy, and by quantitative real time PCR analysis of atrial natriuretic peptide (Nppa), periostin (Postn), connective tissue growth factor (Ctgf) and β-myosin heavy chain (Myh7). Our data show that a chronic treatment with the β(2)-AR agonist terbutaline at low antiallodynic dose does not affect cardiovascular parameters, whereas the mixed β(1)/β(2)-AR agonist isoproterenol induces cardiac hypertrophy. These data suggest that low doses of β(2)-AR agonists may provide a suitable treatment with rare side effects in neuropathic pain management. This study conducted in an animal model requires clinical confirmation in humans.     

Stereoselectivity at the β2-adrenoceptor on macrophages is a major determinant of the anti-inflammatory effects of β2-agonists

Previous research has shown that beta-adrenoceptor (beta-AR) agonists have potent anti-inflammatory capabilities, e.g. represented by suppression of release of the proinflammatory cytokines. Aim of this research was to determine whether the effects of beta-agonists on LPS-induced TNFalpha and IL-10 release are influenced by their different stereochemistry. In addition, the role of the beta-AR subtypes was studied. The effect of two stereoisomers of the selective beta2-AR agonist TA2005 [(R,R)- and (S,S)-] on the LPS-induced TNFalpha and IL-10 release by U937 macrophages was compared. The (R,R)-stereoisomer was 277 times more potent in inhibiting the TNFalpha release than the (S,S)-form. The (R,R)-stereoisomer also appeared to be more potent in increasing the IL-10 release. In radioligand binding studies the affinity of (R,R)-TA2005 for the beta-adrenoceptor was 755 times higher than the (S,S)-TA2005 stereoisomer. In addition, the elevation of intracellular cAMP in U937 cells appeared to be stereoselective: (R,R)-TA2005 was more potent in elevating intracellular cAMP. The effect of both stereoisomers on the LPS-induced TNFalpha release could almost completely be antagonized by preincubation with the selective beta2-AR-antagonist ICI-118551. Further evidence that the effect of the beta-agonists is mediated via the beta2-adrenoceptor subtype exclusively was acquired by incubation of U937 cells with selective beta1- and beta3-agonists. None of these receptor subtype agonists showed significant suppressive effect on TNFalpha release. This study provides additional proof that the anti-inflammatory effects of beta2-agonists are mediated via the beta2-adrenoceptor and indicates that these effects are highly dependent on the stereoselectivity of the ligand.