• association rate (kon);
  • dissociation rate (koff);
  • competition binding;
  • equilibrium;
  • kinetics;
  • adrenoceptor;
  • agonist;
  • intrinsic activity


  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conflict of interest
  8. References

BACKGROUND AND PURPOSE β2-Adrenoceptor agonists are important bronchodilators used for the treatment of chronic obstructive pulmonary disease and asthma. Clinical data on β2-adrenoceptor agonists show a range of onset and duration of action. We have investigated whether the receptor binding kinetics of β2-adrenoceptor agonists can explain their observed onset of action and duration of effect in the clinic.

EXPERIMENTAL APPROACH [3H]-DHA was used to label β2-adrenoceptors expressed in CHO-cell membranes (Kd of 0.084 nM). Competition kinetic experiments were performed in the presence of unlabelled β2 agonists at 37°C in HBSS containing GTP. To determine the kinetic parameters, three concentrations (10, 3 and 1 ×Ki) of the unlabelled compound were employed against a fixed concentration of [3H]-DHA (0.6 nM).

KEY RESULTS The clinically used β2-adrenoceptor agonists exhibited a range of association and dissociation rates. The kinetic Kd and the competition Ki values of the eight β2-adrenoceptor agonists examined were strongly correlated, suggesting that the method had produced accurate koff and kon rates. The kinetic on-rate was highly correlated with equilibrium binding affinity.

CONCLUSIONS AND IMPLICATIONS Although the β2-adrenoceptor agonists displayed a range of kinetic rate parameters, simulations at relevant drug concentrations suggest that receptor kinetics do not play an important role in determining onset of action in the clinic. In addition, it is unlikely that receptor kinetics exert an important influence on the duration of action of these agonists, as indacaterol (once daily dosing) had a shorter residency time at the receptor than salmeterol (twice daily dosing).


1-[4,6-propyl-3H] dihydroalprenolol


non-specific binding




chronic obstructive pulmonary disease


long-acting β2-adrenoceptor agonist


  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conflict of interest
  8. References

Long-acting β2-adrenoceptor agonists (LABAs) play an important role in the treatment of asthma and chronic obstructive pulmonary disease (COPD), providing improved symptom control. One aspect of the biology of LABAs which remains unresolved is the factors which determine their duration of action. Duration of action often depends on many pharmacokinetic factors, including absorption, distribution and clearance (Smith et al., 1996). However, the direct kinetics of drug receptor interaction can also play a significant role in drug duration (Copeland et al., 2006).

Of the clinically approved bronchodilators, only indacaterol (150 µg) achieves a 24 h duration of action in COPD patients (Beier et al., 2006; Beeh and Beier, 2010). In contrast, formoterol (12 µg) and salmeterol (50 µg) require twice daily dosing (Wegener et al., 1992; van Noord et al., 1996; Palmqvist et al., 1997; Sutherland et al., 2009), while salbutamol (200 µg) must be given up to four times a day in order to achieve a clinically useful effect (Tashkin and Fabbri, 2010).

It is widely accepted that these agonists have the capacity to ‘reassert’ airway smooth muscle relaxation in vitro despite repeat washing of isolated tissue (Anderson et al., 1994). The retention and reassertion of salmeterol in tissue have been attributed to binding of its aliphatic tail to a so-called ‘exosite’ or ‘exoceptor’, a site distinct from the β2 adrenoceptor, allowing the active saligenin head structure to freely angle on and off the receptor (Johnson, 1992). However, it is now becoming clear that the persistent in vitro relaxant activity and reassertion effect are properties common to several lipophilic β2 agonists that do not possess a long aliphatic side chain (Summerhill et al., 2008), questioning the validity of the exosite model. Perhaps a more compelling model is the plasmalemma diffusion microkinetic theory, in which the plasmalemma lipid bilayer of airway smooth muscle acts as a depot for β2 adrenoceptor agonists with moderate to high lipophilicity (Anderson et al., 1994).

Another potential factor that might influence the duration of action of β2-adrenoceptor agonists is the kinetics of agonist–receptor dissociation. It has been demonstrated that slow dissociation kinetics plays an important role in the duration of drug action of inhaled muscarinic antagonists (Disse et al., 1999). In the case of β-adrenoceptors, several slowly dissociating antagonists from this receptor have been described (Lucas et al., 1979; De Blasi et al., 1988; Pauwels et al., 1988; Keith et al., 1989; Doggrell, 1990; Deyrup et al., 1999). In keeping with their slow dissociation, several of these antagonists, including bornaprolol (FM 24) and ICI 147,798, have been shown to have a relatively long duration of action in vivo that is independent of their plasma levels (Le Fur et al., 1980; Keith et al., 1989).

The long duration of action of the β2-adrenoceptor agonist carmoterol (CHF-4226, TA-2005) has been attributed to its ‘slow dissociation’ from the receptor (Voss et al., 1992). However, this claim is based on in vitro‘wash out’ experiments which do not directly measure the dissociation kinetics of the molecule as it may also be influenced by membrane interactions and drug ‘rebinding’ (Vauquelin and Charlton, 2010).

In addition to a long duration of action, a fast onset of action is a desirable property of inhaled β2-adrenoceptor agonists, providing rapid relief of symptoms. Fast-acting β2-adrenoceptor agonists such as indacaterol, formoterol and salbutamol can produce bronchodilation within 1–5 min (Brookman et al., 2007; Van Noord et al., 1998), whereas the slower-acting β2-adrenoceptor agonist salmeterol can take between 6 and 30 min to produce a significant bronchodilatory effect (Palmqvist et al., 1997; Brookman et al., 2007). The kinetics of drug–receptor interaction could be one factor important in determining the onset and subsequent relief of symptoms, as receptor kinetics will determine the initial rate of receptor occupancy. A recent review by Tashkin and Fabbri, (2010) details the onset and duration of action of therapies currently used to treat COPD.

The aim of this work was to investigate the kinetic properties of several clinically relevant β2-adrenoceptor agonists with widely varying onset and durations of action to determine if any relationships exist. The association and dissociation rates of compounds are traditionally assessed directly by monitoring the specific binding of a labelled form (often radiolabelled) of the ligand of interest. Motulsky and Mahan (1984) have previously described a method to quantify the kinetic parameters of unlabelled compounds. The practical application of this method was demonstrated by Dowling and Charlton (2006), and more recently we have used this technique to explore the kinetics of muscarinic M3 receptor agonists (Sykes et al., 2009). In brief, a kinetically characterized radioligand is added simultaneously with an unlabelled ligand to the receptor preparation of interest. The experimentally derived rate of specific radioligand binding can then be modelled to provide the association and dissociation rates of the unlabelled compound.

Kinetic competition models rely on the radiolabel having a rapid enough off-rate such that the competing ligand is able to reach equilibrium with the receptor in the time frame of the experiment. We have characterized two commercially available radiolabels, the commonly used β2-adrenoceptor radiolabel [125I]-CYP and the less widely used [3H]-DHA. Following these initial exploratory binding studies, we selected [3H]-DHA as the most suitable ligand for determining the kinetic properties of our unlabelled β2-adrenoceptor agonists.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conflict of interest
  8. References

Cell culture

CHO cells stably transfected with the human β2-adrenoceptor were grown adherently in Ham's F-12 Nutrient Mix GlutaMAX-1, containing 10% fetal calf serum, and 0.5 mg·mL−1 Geneticin (G-418). Cells were maitained at 37°C in 5% CO2/humidified air. Cells were routinely subcultured at a ratio between 1:10 and 1:20 twice weekly using tryspin-EDTA to lift cells.

Cell membrane preparation

CHO cells expressing the human β2-adrenoceptor were grown to 80–90% confluency in 500 cm2 cell culture plates at 37°C in 5% CO2. All subsequent steps were conducted at 4°C to avoid receptor degradation. The cell culture media were removed, and ice-cold buffer [1 × 10 ml; 10 mM HEPES, 0.9% (w/v) NaCl, and 0.2% (w/v) EDTA, pH 7.4] was added to the cells, which were then scraped from the plates into a 50-ml Corning tube (Corning Inc., Corning, NY, USA) and subsequently centrifuged at 250 g for 5 min to allow a pellet to form. The supernatant fraction was aspirated, and 10 ml per 500-cm2 tray of wash buffer (10 mM HEPES and 10 mM EDTA, pH 7.4) was added to the pellet. This was homogenized using an electrical homogenizer Ultra-Turrax (Ika-Werk GmbH & Co. KG, Staufen, Germany) (position 6, 4 × 5-s bursts) and subsequently centrifuged at 48,000 g at 4°C (Beckman Avanti J-251 Ultracentrifuge; Beckman Coulter, Fullerton, CA, USA) for 30 min. The supernatant was discarded, and the pellet was rehomogenized and centrifuged as described above in wash buffer. The final pellet was suspended in ice-cold 10 mM HEPES and 0.1 mM EDTA, pH 7.4, at a concentration of 5 to 10 mg/ml. Protein concentration was determined by the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA) based on the method of Bradford (1976) using BSA as a standard, and aliquots were maintained at −80°C until required.

Common procedures applicable to all radioligand binding experiments

All radioligand experiments were conducted in 96-deep well plates, in assay binding buffer, HBSS pH 7.4 containing 0.1% BSA, 0.01% ascorbic acid and 100 µM GTP. In all cases, non-specific binding (NSB) was determined in the presence of 1 µM propranolol. After the indicated incubation period, bound and free radiolabels were separated by rapid vacuum filtration using a FilterMateTM Cell Harvester (PerkinElmer Life and Analytical Sciences, Beaconsfield, UK) onto 96 well GF/B filter plates previously coated with 0.5% (w/v) polyethylenimine and rapidly washed three times with ice-cold 75 mM HEPES, pH 7.4. After drying (>4 h), 40 µL of MicroscintTM 20 (PerkinElmer Life and Analytical Sciences) was added to each well and radioactivity was quantified using single photon counting on a TopCountTM microplate scintillation counter (PerkinElmer Life and Analytical Sciences). Aliquots of radiolabel were also quantified accurately to determine how much radioactivity was added to each well using liquid scintillation spectrometry on LS 6500 scintillation counter (Beckman Coulter, High Wycombe, UK). In all experiments, total binding never exceeded more than 10% of that added, limiting complications associated with depletion of the free radioligand concentration (Carter et al., 2007).

Saturation binding studies

CHO cell membranes containing the β2-adrenoceptor were incubated in 96-deep well plates at 37°C in assay binding buffer with a range of concentrations of [125I]-CYP (∼200–0.05 pM) and [3H]-DHA (∼3–0.001 nM) at 3 and 15 µg per well respectively, for 180 min with gentle agitation to ensure equilibrium was reached. Saturation binding was performed in a final assay volume of to 1.5 mL to avoid significant ligand depletion.

Determination of the association rate (kon) and dissociation rate (koff) of [125I]-CYP and [3H]-DHA

To accurately determine kon and koff values, the observed rate of association (kob) was calculated at least three different concentrations of either [125I]-CYP or [3H]-DHA. The appropriate concentration of radioligand was incubated with β2-adrenoceptor CHO cell membranes (3 and 15 µg· per well) in assay binding buffer with gentle agitation (final assay volume 1000 µL). Exact concentrations were calculated in each experiment by liquid scintillation counting. Free radioligand was separated by rapid filtration at multiple time points to construct association kinetic curves as described previously by Sykes et al. (2009). The resulting data were globally fitted to the association kinetic model to derive a single best fit estimate for kon and koff as described under Data analysis.

Determination of agonist affinity constants (Ki)

To obtain affinity estimates of unlabelled agonists, [3H]-DHA competition experiments were performed at equilibrium. [3H]-DHA was used at a concentration of approximately 0.6 nM (∼25 000 c.p.m. final assay volume of 0.5 mL), such that the total binding never exceeded more than 10% of that added. Radioligand was incubated in the presence of the indicated concentration of unlabelled agonist and CHO cell membranes (15 µg· per well) at 37°C, with gentle agitation for 180 min.

Competition binding kinetics

The kinetic parameters of unlabelled agonists were assessed using a competition kinetic binding assay as described by Sykes et al. (2009). This approach involves the simultaneous addition of both radioligand and competitor to receptor preparation, so that at t = 0 all receptors are unoccupied. Approximately 0.6 nM [3H]-DHA (a concentration which avoids ligand depletion in this assay volume) was added simultaneously with the unlabelled compound (at t = 0) to CHO cell membranes containing the human β2-adrenoceptor (15 µg· per well) in 500 µL assay buffer. The degree of [3H]-DHA bound to the receptor was assessed at several time points by filtration harvesting and liquid scintillation counting, as described previously. NSB was determined as the amount of radioactivity bound to the filters and membrane in the presence of propranolol (1 µM) and was subtracted from each time point, meaning that t = 0 was always equal to 0. Each time point was conducted on the same 96-deep well plate incubated at 37°C with constant agitation. Reactions were considered stopped once the membranes reached the filter, and the first wash was applied within 1 s. Three different concentrations of unlabelled competitor were tested to ensure that the rate parameters calculated were independent of ligand concentration. All compounds were tested at one-, three- and 10-fold their respective Ki and data were globally fitted using Equation 3 to simultaneously calculate kon and koff.

Data analysis and statistical procedures

As the amount of radioactivity varied slightly for each experiment (<5%), data are shown graphically as the mean ± range for individual representative experiments, whereas all values reported in the text and tables are mean ± SEM for the indicated number of experiments unless otherwise stated. All experiments were analysed by either Deming regression or non-linear regression using Prism 4.0 (GraphPad Software, San Diego, CA, USA).

Competition binding.  Competition displacement binding data were fitted to sigmoidal (variable slope) curves using a four-parameter logistic equation:

  • image(1)

IC50 values obtained from the inhibition curves were converted to Ki values using the method of Cheng and Prusoff (1973). Equation 1 was utilized for data presented in Figure 3.

Association binding.  [125I]-CYP and [3H]-DHA association data were globally fitted to the following equation, where L is the concentration of radioligand in nM using GraphPad Prism 4.0 to determine a best fit estimate for kon and koff. Equation 2 was utilized for data presented in Figure 2A,B.

  • image(2)

Competition kinetic binding.  Association and dissociation rates for unlabelled agonists were calculated using the equations described by Motulsky and Mahan (1984) using a global fitting model:

  • image
  • image
  • image
  • image
  • image
  • image
  • image
  • image(3)

where X is time (min), Y is specific binding (c.p.m.), k1 is kon[3H]-DHA, k2 is koff[3H]-DHA, L is the concentration of [3H]-DHA used (nM) and I is the concentration of unlabelled agonist (nM). Fixing the above parameters allowed the following to be simultaneously calculated: Bmax is total binding (c.p.m.), k3 is association rate of unlabeled ligand (M−1 min−1) or kon, and k4 is the dissociation rate of unlabelled ligand (min−1) or koff. Equation 3 was utilized for data presented in Figure 4A–H.

Simulations.  The observed association of ligand to receptor (kob) (see Figure 7A–C) was simulated in Prism 4.0 using Equation 2. Fixed kinetic parameters (k1–2) for the ligands determined in the competition kinetic studies were used to simulate the binding of ligand to receptor over time, at the concentration of ligand required to occupy 50% of available receptors (Kd), or at an EC25 concentration equivalent to 25% of the maximal cAMP response to isoprenaline (values taken from Battram et al., 2006). In addition, simulations were performed at relative clinical doses. In the clinic, salmeterol, indacaterol, salbutamol and formoterol are dosed at 50, 150, 200 and 12 µg respectively. A concentration of salmeterol at 10-fold its Kd (7.5 nM) was chosen for modelling purposes and concentrations of indacaterol, formoterol and salbutamol were calculated based on their clinical doses relative to this concentration of salmeterol. For example, doses of salbutamol and indacaterol are four and threefold higher than that of salmeterol, so concentrations of 22.5 nM and 30 nM were used for modelling purposes. In contrast, formoterol is given at a 4.17-fold lower dose than salmeterol, so a lower relative concentration of 1.8 nM was used. Dissociation rates for the β2 agonists were modelled in Prism 4.0 using Equation 4 (Figure 7D).

  • image(4)


1-[4,6-propyl-3H]dihydroalprenolol ([3H]-DHA specific activity 91 Ci·mmol−1) was obtained from Amersham Biosciences UK Ltd. (GE Healthcare, Chalfont St Giles, UK) and [125I]-iodo-(-)-cyanopindolol ([125I]-CYP specific activity 2200 Ci·mmol−1) was obtained from PerkinElmer. 96-deep well plates and 500 cm2 cell culture plates were purchased from Fisher Scientific (Loughborough, UK). 96-well GF/B filter plates were purchased from Millipore (Watford, UK). Sodium bicarbonate, ascorbic acid, EDTA, sodium chloride, GTP, propranolol (-)isoprenaline hydrochloride, formoterol fumarate, and (-)adrenaline were obtained from Sigma Chemical Co Ltd. (Poole, UK). Salmeterol and salbutamol hemisulfate were obtained from Tocris Cookson Inc. (Bristol, UK). A related sulfonamide analogue of salmeterol 3-[4-[[6-[[(2R)-2-hydroxy-2-[4-hydroxy-3 (hydroxymethyl)phenyl]ethyl]amino]hexyl] oxy]butyl]benzenesulfonamide (Compound 1; Procopiou et al., 2009; Rosethorne et al., 2010) and indacaterol were synthesized by Global Discovery Chemistry (Novartis, Horsham, UK). All cell culture reagents including HBSS and HEPES were purchased from Gibco (Invitrogen, Paisley, UK).


  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conflict of interest
  8. References

Characterization of [125I]-CYP and [3H]-DHA saturation binding

Specific [125I]-CYP and [3H]-DHA binding to human β2-adrenoceptors expressed in CHO membranes was saturable and best described by the interaction of each radioligand with a single population of high-affinity binding sites. The expression level of the human β2-adrenoceptor recombinantly expressed in CHO cells was assessed, using [125I]-CYP saturation binding, as 499 ± 129 fmol·mg−1 protein (Figure 1A). A similar value of 529 ± 16 fmol·mg−1 protein was obtained when saturation binding was carried out with [3H]-DHA (Figure 1B). From these studies, the equilibrium dissociation constant (Kd) of [125I]-CYP and [3H]-DHA was determined to be 4.10 ± 0.93 pM and 83.5 ± 11.1 pM respectively.


Figure 1. Saturation analysis of the binding of (A) [125I]-CYP and (B) [3H]-DHA to CHO membranes expressing the human β2-adrenoceptor. CHO-β2 cell membranes (3 and 15 µg· per well respectively) were incubated for 180 min with gentle agitation with increasing concentrations of radiolabel. NSB was defined by 1 µM propranolol. Specific binding is presented as the mean from a representative of three experiments performed in duplicate.

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Characterization of [125I]-CYP and [3H]-DHA kinetic parameters

In order to establish a suitably robust system for determining the kinetic parameters of unlabelled β2-adrenoceptor agonists, a comparison was made of two commercially available antagonist radiolabels [3H]-DHA and [125I]-CYP in HBSS at 37°C. The observed association rate of a ligand is in part dependent upon the concentration of radiolabel used, so we constructed a family of association curves using a range of [125I]-CYP (∼250–10 pM) and [3H]-DHA concentrations (∼0.6–0.1 nM) concentrations.

Each association curve was monitored to equilibrium, the point at which no further binding was observed (Figure 2A,B). Binding followed a simple law of mass action model, kob increasing in a linear manner with radioligand concentration (data not shown). Consequently, [125I]-CYP and [3H]-DHA association data were globally fitted to derive a single best fit estimate for kon and koff of each radioligand. Kinetic kon and koff values determined for [125I]-CYP and [3H]-DHA are shown in Table 1. Association rates for both ligands were similar; however, there was a 10-fold difference in their dissociation rates, with [125I]-CYP being considerably slower than [3H]-DHA to dissociate (0.0056 vs. 0.083 min−1). This difference in dissociation rate is largely responsible for the difference in the speed at which these two ligands reach equilibrium with the receptor. The kinetically derived Kd value for [3H]-DHA was in good agreement with the Kd estimated from the saturation analysis (33.9 ± 12.3 vs. 83.5 ± 11.1 pM), as was that for [125I]-CYP (1.6 ± 0.2 vs. 4.10 ± 0.93 pM). The small differences between the two approaches are most likely because the incubation time for the saturation studies (3 h, limited by membrane stability) was insufficient to achieve full equilibrium between receptor and radioligand.


Figure 2. Kinetics of the interaction of [125I]-CYP and [3H]-DHA with CHO membranes expressing the human β2-adrenoceptor. The kon and koff values for (A) [125I]-CYP and (B) [3H]-DHA were determined by incubating CHO-β2 cell membranes (3 and 15 µg· per well, respectively) with the indicated concentrations of [125I]-CYP and [3H]-DHA for various time periods. Association data were fitted to a global fitting model using GraphPad Prism 4.0 to simultaneously calculate kon and koff. Data are presented as the mean ± range from a representative of ≥3 experiments performed in duplicate.

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Table 1.  Kinetic binding parameters and affinity values of [3H]-DHA and [125I]-CYP for human β2-adrenoceptor receptors
RadiolabelKinetic Kd (pM)kon (M−1 min−1)koff (min−1)Dissociation half-life t1/2 (min)Saturation Kd (pM)
  1. Data are mean ± SEM for ≥3 experiments performed in duplicate.

[3H]-DHA33.9 ± 12.32.86 ± 0.32 × 1090.083 ± 0.0208.483.5 ± 11.1
[125I]-CYP1.6 ± 0.23.68 ± 0.32 × 1090.0056 ± 0.0001123.74.1 ± 0.9

Kinetic observations are accurate only if the competition kinetic curves are allowed to approach equilibrium. The slower the off-rate of the radioligand from its receptor, the longer the time taken to reach equilibrium not only for kinetic determinations but also for equilibrium competition experiments. A faster dissociating radioligand ensures that the total incubation period of the assays is reduced; which is an important practical consideration. In preliminary experiments, inadequate competition was observed between CYP and the competing agonists, thus competition kinetic parameters were determined using DHA which has a relatively faster dissociation rate.

[3H]-DHA equilibrium competition binding

The β2-adrenoceptor binding profile of the agonists was determined in buffer containing GTP (0.1 mM) to ensure that agonist binding only occurred to the uncoupled form of the β2 receptor. Each of the β2 agonist ligands produced concentration-dependent inhibition of the specific binding of [3H]-DHA to sites on CHO-β2-adrenoceptor membranes. Examples of competition data are shown in Figure 3. Equilibrium competition binding data were fitted to a four-parameter logistic equation to obtain pIC50 and Hill slope parameter estimates. Equilibrium dissociation constants (pKi) were subsequently determined from pIC50 values using the Cheng and Prusoff equation (Cheng and Prusoff 1973). The binding affinity of the β2-adrenoceptor agonists for the β2-adrenoceptor is shown in Table 2.


Figure 3. Competition between [3H]-DHA and increasing concentrations of isoprenaline, salmeterol, salbutamol, formoterol, indacaterol, adrenaline, Compound 1 and carmoterol for human β2-adrenoceptors expressed in the CHO cells in the presence of GTP. Membranes (15 µg· per well) from CHO-β2 cells were incubated in HBSS containing 0.1 mM GTP at 37°C (as described in Methods) with 0.6 nM [3H]-DHA and the indicated concentrations of competitor for 180 min. Data are presented as the mean ± range from a representative of ≥3 experiments performed in duplicate.

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Table 2.  Affinity values and kinetically derived parameters for unlabelled ligands
Compoundkon (M−1min−1)koff (min−1)t1/2 (min)pKd (koff/kon)pKi
  1. Data are mean ± SEM for ≥3 experiments performed in duplicate.

Isoprenaline2.47 ± 1.39 × 1073.06 ± 1.530.236.89 ± 0.086.73 ± 0.15
Salmeterol4.31 ± 1.34 × 1090.76 ± 0.060.919.70 ± 0.209.19 ± 0.07
Salbutamol2.05 ± 1.03 × 1074.06 ± ± 0.146.54 ± 0.07
Formoterol2.15 ± 0.45 × 1083.29 ± 0.790.217.83 ± 0.097.28 ± 0.10
Indacaterol8.74 ± 2.12 × 1073.48 ± 0.420.207.37 ± 0.117.04 ± 0.05
Adrenaline3.15 ± 0.61 × 1065.12 ± 1.390.145.85 ± 0.105.82 ± 0.05
Compound 13.25 ± 1.7 × 1080.41 ± 0.041.698.79 ± 0.228.80 ± 0.08
Carmoterol8.66 ± 0.46 × 1070.46 ± 0.091.518.32 ± 0.118.16 ± 0.11

Competition kinetic binding

The association and dissociation rates of [3H]-DHA were determined in each experimental run and these values were used to calculate the kon (k3) and koff (k4) of the unlabelled compound using Equation 3, as detailed in Methods. Representative curves for the β2-adrenoceptor agonists tested are shown in Figure 4A–H. To ensure that each ligand displayed classical competitive and reversible binding, each agonist was assayed at three different concentrations, one-, three- and 10-fold Ki.


Figure 4. [3H]-DHA competition kinetic curves in the presence of isoprenaline (A), salmeterol (B), salbutamol (C), formoterol (D), indacaterol (E), adrenaline (F), Compound 1 (G) and carmoterol (H). CHO-β2 membranes were incubated with ∼0.6 nM [3H]-DHA and either 0-, 1-, 3 or 10-fold Ki. Plates were incubated at 37°C for the indicated time points and NSB levels were determined in the presence of 1 µM propranolol. Data were fitted to the equations described in the Methods to calculate kon and koff values for the unlabelled agonists; these are summarized in Table 2. Data are presented as mean ± range from a representative of ≥3 experiments performed in duplicate.

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The pattern of [3H]-DHA binding over time was dependent upon the off-rate of the competing agonist. [3H]-DHA association in the presence of more slowly equilibrating competitors was bi-phasic. Progression curves for [3H]-DHA alone and in the presence of three different concentrations of competitor were globally fitted to Equation 3, enabling the calculation of both kon (k3) and koff (k4) for each of the agonists, as reported in Table 2. As the koff values determined were similar across the cohort, we tested whether the data were sufficient to discriminate between the agonists. The quality of fit was worse when the koff was fixed to any value outside that predicted by simultaneous fitting. To validate the rate constants, the kinetically derived Kd values (koff/kon) were compared with the affinity constant (Ki) obtained from equilibrium competition binding experiments (Figure 5). There was a very good correlation (r2= 0.97, P < 0.0001) between these two values.


Figure 5. Correlation between pKi and kinetically derived pKd for the eight test agonists. pKi values were taken from [3H]-DHA competition binding experiments at equilibrium. The values comprising the kinetically derived Kd (koff/kon) were taken from the experiments shown in Figure 4. Data are presented as mean ± SEM from three or more experiments.

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A relationship between koff and competition-derived pKi has been suggested previously for β2-adrenoceptor antagonists (Affolter et al., 1985; Contreras et al., 1986). A correlation plot of log koff versus kinetically derived pKd for the eight agonists tested in this study also revealed a significant correlation (Figure 6A, r2= 0.64, P < 0.05). Interestingly, there was a more highly significant correlation between log kon and pKd values (Figure 6B, r2= 0.92, P < 0.0002) than that achieved for koff. These data imply that kon plays a more important role in defining the equilibrium dissociation constant of β2-adrenoceptor agonists.


Figure 6. Correlation of pKi with log koff (A) and log kon (B) determined for the eight test agonists. pKi values were taken from [3H]-DHA competition binding experiments. All data used in these plots are detailed in Table 2. Data are presented as mean ± SEM from three or more experiments.

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Using kinetic parameters to model the rate of agonist occupancy and dissociation from the receptor

The rate of receptor occupancy is one factor which could potentially play a significant role in the rate of onset of the actions of β2-adrenoceptor agonists. When kob was simulated at Kd concentration, salmeterol had a slower rate of receptor occupancy than the other clinically used ligands tested (Figure 7A). Comparing agonists at the same level of receptor occupancy does not, however, account for any differences in potency that are a consequence of different intrinsic efficacies (Charlton, 2009). To address this, the rate of agonist association was simulated using agonist concentrations based on potency from in vitro experiments (equivalent to 25% of the maximal cAMP response to isoprenaline, data taken from Battram et al., 2006). Under these conditions, there were no clear differences in the rate of association of the four clinically used β2-adrenoceptor ligands, suggesting that binding rate is not a key determinant of clinical onset of action (Figure 7B).


Figure 7. Modelling the association and dissociation of clinically relevant LABAs. Simulated binding of clinically relevant LABAs salmeterol, indacaterol, formoterol, salbutamol and carmoterol to human β2-adrenoceptors, at (A) Kd concentrations (see Table 2 for details), (B) EC25 concentrations salmeterol (0.79 nM), indacaterol (3.2 nM), formoterol (0.6 nM) and salbutamol (178 nM), (EC25 values from Battram et al., 2006) and (C) concentrations derived from relative clinical doses. For simulation purposes salmeterol at a concentration 10× its Kd (7.5 nM) was chosen and concentrations of indacaterol (22.5 nM), formoterol (1.8 nM) and salbutamol (30 nM) were calculated based on their clinical doses (see Results section) relative to this concentration of salmeterol. (D) Stimulated dissociation rates of clinically relevant β2 ligands from β2-adrenoceptors based on off-rates determined in competition kinetic binding experiments. All parameters derived from these plots are detailed in Table 3.

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Although comparing in vitro potency data provides more relevant simulations than comparing agonist occupancy, it is still far removed from the relative doses of each compound used in the clinic. Ideally, clinical pharmacokinetic (PK) data are used to develop a pharmacokinetic /pharmacodynamic (PK/PD) model but, for inhaled compounds, plasma levels do not reflect the pharmacodynamically relevant concentrations, but rather the spillover of drug from the effect compartment. In the absence of direct PK measurements in the lung, we have considered the clinical doses of the compounds administered by inhalation and simulated receptor association rates at relative concentrations of the compounds assuming complete dissolution following dosing.

In the clinic, salmeterol, indacaterol, salbutamol and formoterol are given at doses of 50, 150, 200 and 12 µg respectively. Figure 7C models the rate of receptor occupancy of these agonists at concentrations based on the relative clinical doses of the four drugs. A concentration of salmeterol at 10-fold its Kd (7.5 nM) was chosen for modelling purposes, and concentrations of indacaterol, formoterol and salbutamol were calculated based on their clinical doses relative to this concentration of salmeterol. For example, salbutamol and indacaterol are dosed four- and threefold higher than salmeterol, so concentrations of 22.5 nM and 30 nM were used for modelling purposes. In contrast, formoterol is dosed 4.17-fold lower than salmeterol so a lower relative concentration of 1.8 nM was used. Under these conditions, salmeterol occupies the receptors more rapidly than the other agonists, reinforcing the conclusion that receptor kinetics does not have an important influence on onset of action. As observed in the previous simulation, salmeterol occupies a far larger proportion of receptors than the other agonists when their clinical doses are compared. Indacaterol has the longest duration of action in the clinic (>24 h) of all the ligands tested in this study, with salbutamol having the shortest duration of action at 4–6 h (Brookman et al., 2007). When the relationship between receptor occupancy and dissociation for all four ligands was simulated in Figure 7D, all were fully dissociated within 10 min, suggesting that dissociation rate has little or no role to play in the duration of action of clinically used long-acting β2 ligands. Dissociation t½ values and kob values from these simulations are detailed in Table 3.

Table 3.  Simulated t½ and kob values for clinically relevant β2-adrenoceptor agonists determined at binding Kd concentration, CHO-β2 cAMP assay derived EC25 concentration and concentrations derived from relative clinical doses (see Figure 7 for graphical representation)
CompoundDissociationBinding assay –Kd concentrationcAMP assay – EC25 concentrationConcentration relative to clinical dose
t½ (min)kob (min−1)% receptor occupiedkob (min−1)% receptor occupiedkob (min−1)% receptor occupied
  1. ND, not determined.



  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conflict of interest
  8. References

The aim of the present study was to measure the kinetic properties of a series of β2-adrenoceptor agonists to determine if the kinetic rate constants influenced the observed onset of action and duration of effect in the clinic. Direct kinetic analysis of the binding of ligands to β2-adrenoceptors has been limited primarily to the study of the interactions of antagonists with the receptor. Only a few kinetic studies with radiolabelled agonist have been performed due to the limited number of radiolabelled agonists available. In addition, the studies with agonists have largely been performed in the absence of GTP, thus molecular interpretation is likely to be complicated by interactions between the receptor and its associated G protein. Kinetic parameters for isoprenaline have been determined previously in a competition kinetic format but at non-physiological temperature (–10°C) designed to slow ligand dissociation (Contreras et al., 1986). To our knowledge, the current study is the first time that kinetic rate constants have been derived for β2-adrenoceptor agonists in a binding assay performed at physiological temperature and sodium concentrations in the presence of guanine nucleotide. These studies were designed to more closely mimic in vivo conditions, allowing better extrapolation to the clinical situation. Indirect isoprenaline koff measurements have been made previously in HBSS using the rate of cAMP decline following the addition of a high concentration of propranolol, as a measure of koff (Deyrup et al., 1999). The value of 5.5 min−1 was in agreement with the reported koff (4 min−1) for isoprenaline based on recovery of a chemo attractant-mediated cellular response inhibited by β2-adrenoceptor activation in neutrophils (Mueller et al., 1988). More recently, the interaction of ligands with purified β2-adrenoceptors has been measured by plasmon waveguide resonance spectroscopy. Using this method, isoprenaline and adrenaline produced off-rates of 4.68 min−1 and 7.20 min−1, respectively, following displacement by alprenolol (Devanathan et al., 2004). These values, as well as the values obtained for isoprenaline in functional studies (Mueller et al., 1988; Deyrup et al., 1999), agree closely with the value of 3.06 min−1 for isoprenaline and 5.12 min−1 for adrenaline obtained in our competition kinetic studies.

The values for koff for the binding of agonists correlated with the equilibrium dissociation constant (r2= 0.64, P < 0.05), which is in agreement with previous studies suggesting that affinity and kinetic off-rate are linked (Affolter et al., 1985; Contreras et al., 1986 and Deyrup et al., 1999). However, the fastest and the slowest dissociating ligands tested in this study differed in koff only by a factor of 10-fold.

The association rate constant, kon, is thought to depend largely on diffusion (Weiland and Molinoff, 1981). Interestingly, kon values for the binding of β2-adrenoceptor agonists have been shown to be consistently lower when compared with β2-adrenoceptor antagonists when determined at −10°C (Contreras et al., 1986). This finding is thought to be the result of isomerization of the receptor on binding of the agonist. Our data obtained at physiological temperature would seem to support this theory as [3H]-DHA and [125I]-CYP have very fast on-rates, compared with the β2-adrenoceptor agonists with high intrinsic activity such as adrenaline. There was, however, a much larger difference in kon values between the β2 agonists. These ranged from 4.31 × 109 M−1 min−1 for the partial agonist salmeterol to 3.15 × 106 M−1 min−1 for the full agonist adrenaline. It is tempting to speculate that the rapid on-rate for the partial agonist salmeterol compared with the full agonist adrenaline (>1000-fold difference) is due to a reduction in the magnitude of the isomerization step it undergoes when activating the receptor. Interestingly, kon correlated better with the Kd than koff values, suggesting that the on-rate is more important for determining the equilibrium affinity of these agonists than the off-rate. We have previously observed this phenomenon in a study of muscarinic M3 agonist kinetics (Sykes et al., 2009).

Our simulations would suggest that the kinetic rate parameters determined for the clinically used β2-adrenoceptor ligands are unlikely to play a significant role in determining the duration of action of these molecules. For example, salbutamol is a short-acting β2 agonist which requires dosing several times a day. However, we have shown that the kinetic off-rate of salbutamol is similar to indacaterol (4.06 vs. 3.48 min−1), which is a long-acting β2-adrenoceptor ligand requiring only once daily administration for a 24 h duration of action in asthma (Kanniess et al., 2006; Beeh et al., 2007; Chuchalin et al., 2007) and COPD patients (Beier et al., 2006, Beeh and Beier, 2010). Salmeterol has a slower off-rate than indacaterol and yet requires twice daily administration (Sutherland et al., 2009). Thus, duration of action and kinetic off-rate do not appear to be linked for the β2-adrenoceptor agonists tested in this study. This is in contrast to a recent study by Casarosa and colleagues (2011) who reported a dissociation half-life for [3H]-olodaterol from the G-protein coupled form of the β2-adrenoceptor of 17.8 h, concluding this contributes to its 24 h duration of action. However, these studies were conducted at non-physiological temperature, in the absence of sodium ions and GTP. This absence of guanine nucleotide in particular means there is likely to be two populations of receptors, a low agonist affinity state that is uncoupled from G-proteins and a high-affinity agonist state that is stabilized by the guanine nucleotide-free Gα subunit in a ternary complex. This ternary complex is stable in a well-washed membrane preparation, but because GTP is present in the cytoplasm at high concentrations, this ternary complex is very short lived in the whole cell (Lemoine, 1992). The stable agonist high-affinity state can therefore be considered as an artefact of the experimental design, meaning it is unlikely that the very slow off-rate of olodaterol observed in this study will be relevant in the clinic. Indeed, the twice daily β2-adrenoceptor [3H]-formoterol has been reported to have a very slow dissociation from this same receptor species, so it is unlikely this property is unique to olodaterol (Lemoine, 1992). It has also been suggested that the long duration of carmoterol observed in vitro in ‘wash out’ experiments can be attributed to its slow dissociation from the β2 receptor (Voss et al., 1992); however, based on its measured kinetic koff (0.46 min−1), we would predict almost complete dissociation of carmoterol from β2 receptors within 5 min. Therefore, the long duration of action of carmoterol is more likely to be attributable to membrane interactions and so-called drug ‘rebinding’ which are inherent features of ‘washout’ experiments (Vauquelin and Charlton, 2010).

Recently, a mathematical model describing the washout of LABAs from the β2-adrenoceptor was reported, but due to the lack of published kinetic binding data, it relied on receptor dissociation values estimated indirectly from functional studies looking at the reversibility of agonist effects (Szczuka et al., 2009), creating debate over the accuracy of the conclusions (Coleman, 2009). The incorporation of our new kinetic parameters into such models would be interesting, although in this particular case the estimated koff value for salmeterol of 0.23 min−1 used by Szczuka et al. in their mathematical simulations was only threefold different to the value determined in our competition binding assay (0.76 min−1).

The clinically used β2-adrenoceptor agonists vary in terms of their onset of action, with salmeterol taking longer to achieve full bronchodilator efficacy than salbutamol, formoterol and indacaterol (Palmqvist et al., 1997; Brookman et al., 2007). To test whether the kinetics of receptor binding was in part responsible for this difference, we simulated receptor association rates using the kinetic parameters measured in this study. Initial simulations at a Kd concentration of each agonist appeared to support this notion, with salmeterol taking longer to reach equilibrium than the other ligands. However, at concentrations that give equivalent functional responses, the onset of action was similar, while at concentrations based on their relative clinical doses, salmeterol occupied the receptors more rapidly than the other agonists. This reinforces the conclusion that receptor kinetics does not have an important influence on onset of action. An interesting observation in these simulations is that salmeterol occupies a far larger proportion of receptors than the other agonists when their clinical doses are compared. This suggests that salmeterol is potentially overdosed relative to the other agonists, perhaps reflecting its poorer efficacy and requirement to bind more receptors to achieve its pharmacological response. In contrast, despite having a relatively low efficacy, salbutamol occupies almost the same proportion of receptors as formoterol (high-efficacy ligand). This suggests that salbutamol is relatively under-dosed in the clinic, which may contribute to the need for four doses in a day to produce a sustained effect on FEV.

In summary, the competition binding studies described here produced accurate kinetic parameters for the binding of a cohort of β2-adrenoceptor agonists to the human β2-adrenoceptor. Although the β2-adrenoceptor agonists exhibited a range of dissociation rates from the receptor, it is doubtful that kinetic rate parameters play a significant role in determining either onset of duration of action. It is more likely that other factors such as lipophilicity (Beattie et al., 2010) and agonist efficacy (Rosethorne et al., 2010) determine the overall onset of action in the clinic, and that partitioning of drug into lipophilic compartments (Anderson et al., 1994; Teschemacher and Lemoine, 1999) after inhalation is the key determinant of their long duration of action.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conflict of interest
  8. References
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