Determination of duration of action of muscarinic m3 antagonists in the electrical field stimulated guinea pig isolated trachea

S. Marshall, J. Marshall, S. Patel, J. Rhodes, E. Stuart and M. Trevethick. Allergy & Respiratory Biology, Pfizer Global R&D, Sandwich, UK

The muscarinic M3 receptor mediating the effects of acetylcholine plays a key role in airway function and disease. The development of antagonists of the M3 receptor such as Ipratropium and the long acting agent Tiotropium have been important in the treatment of chronic obstructive pulmonary disease and asthma (Barnes et al, 1995). This abstract describes a method using the electrical field stimulated (EFS) guinea pig isolated trachea to assess the duration of action of muscarinic M3 antagonists. All compounds are tested at a single concentration of 30 x Ki, determined from ligand binding studies using human M3 receptor recombinantly expressed in CHO cells.

Tracheas were removed from male Dunkin-Hartley guinea pigs (0.45 – 0.55 Kg) culled in a rising concentration of CO2, followed by exsanguination of the vena cava. Tracheal rings (4-5 segments in length) were prepared and then cut directly opposite the muscle layer. The muscle was mounted in the vertical plane in 5ml organ baths in Krebs -Henseleit solution containing indomethacin (3 μM), guanethidine (10 μM) and propranolol (1 μM) at 37 ° C under 1g. wt. initial tension. Krebs was continuously flowed around tissues at 1ml/min. Tissues were stimulated by EFS (0.1ms, 10Hz for 10s every 2 min) at submaximal voltage (10 – 30V). Following 30min equilibration, the flow of Krebs was stopped and single concentrations of standard muscarinic receptor antagonists at 30 x Ki were administered to tissues. Ki values used in this study were determined by ligand binding studies, as described by Rhodes et al (this meeting). Following 2h incubation, compound was removed from tissues by reintroducing the flow of Krebs solution and the duration of action (time to recover 50% of the initial EFS response [T50]) was calculated. Tissues viability was determined at the end of the experiment by a histamine (10 μM) challenge. Statistical analysis was conducted using an unpaired T test.

Atropine, Ipratropium and Tiotropium had Ki values of 1.16nM, 0.96-1.36 (mean, 95%CI, n=7), 0.51nM, 0.48-0.54 (mean, 95%CI, n=5) and 0.09nM, 0.07-0.12 (mean, 95%CI, n=6), respectively. At 30 x Ki, Atropine (30nM), Ipratropium (15nM) and Tiotropium (3nM) inhibited EFS-mediated contractions by 87.9 ± 4.43% (mean ± sem, n=11), 96.4 ± 0.8% and 84 ± 4.16% (mean ± sem, n=6), 2h post administration, respectively. The T50 for Atropine and Ipratropium was 1.61 ± 0.75 h (mean ± sem, n=44) and 1.24 ± 0.34 h (mean ± sem, n=4), respectively, and did not significantly differ from one another (P>0.9). In contrast, the T50 of Tiotropium was >16h (mean, n=6) as all tissues failed to recover their EFS response. At the end of each experiment all tissues contracted in the presence of histamine (10 μM), demonstrating tissue viability. The rank order of duration was Tiotropium > Atropine = Ipratropium.

These results describe a method by which we can rank the duration of action of muscarinic antagonists in the guinea pig trachea, with tissues remaining viable for up to 16h. The method uses predetermined antagonist concentrations determined from a cell based model and a fixed 2h compound incubation period.

Barnes, PJ et al. (1995) Life. Sci., 56 : 853 – 859.