J Pharmacol Sci 115, 374 – 382 (2011) Journal of Pharmacological Sciences © The Japanese Pharmacological Society Full Paper Comparative Characterization of Lung Muscarinic Receptor Binding After Intratracheal Administration of Tiotropium, Ipratropium, and Glycopyrrolate Masaki Ogoda1, Ryo Niiya1, Tadatsura Koshika2, and Shizuo Yamada1,* 1Department of Pharmacokinetics and Pharmacodynamics and Global Center of Excellence (COE) Program, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan 2Pharmacology Research Laboratories, Drug Discovery Research, Astellas Pharma Inc., Tsukuba, Ibaraki 305-8585, Japan Received December 5, 2010; Accepted January 15, 2011 Abstract. The aim of the current study was to characterize comparatively the binding of musca- rinic receptor in the lung of rats intratracheally administered anticholinergic agents (tiotropium, ipratropium, glycopyrrolate) used clinically to treat chronic obstructive pulmonary disease (COPD) and asthma. Binding parameters of [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) were determined in tissues (lung, bladder, submaxillary gland) of rats intratracheally administered tiotropium, ipratropium, and glycopyrrolate. The in vitro binding affinity of tiotropium for the re- ceptors was 10 – 11-fold higher than those of ipratropium and glycopyrrolate. Intratracheal admin- istration of tiotropium (0.6 – 6.4 nmol/kg) caused sustained (lasting at least 24 h) increase in the 3 apparent dissociation constant (Kd) for [ H]NMS binding in rat lung compared with the control value. Concomitantly, there was a long-lasting decrease in the maximal number of binding sites 3 (Bmax) for [ H]NMS. Similary, ipratropium and glycopyrrolate at 7.3 and 7.5 nmol/kg, respectively, 3 brought about a significant increase in Kd for [ H]NMS binding. The effect by ipratropium was observed at 2 h but not 12 h, and that by glycopyrrolate lasted for 24 h. Both agents had little influ- ence on the muscarinic receptors in the bladder and submaxillary gland. The present study provides the first evidence that tiotropium, ipratropium, and glycopyrrolate administered intratracheally in rats selectively bound muscarinic receptors of the lung, and tiotropium and glycopyrrolate had a much longer-lasting effect than ipratropium. Keywords: chronic obstructive pulmonary disease, tiotropium, ipratropium, glycopyrrolate, ex vivo lung muscarinic receptor binding Introduction cholinergic agents. Acetylcholine released from para- sympathetic nerves activates postjunctional muscarinic Chronic obstructive pulmonary disease (COPD) is M3 receptors present in airway smooth muscle to induce characterized by a progressive obstruction of airflow due bronchoconstriction. Since parasympathetic activity is to persistent inflammation of the airways and lung paren- increased in airway inflammation and readily causes the chyma, caused predominantly by chronic cigarette smok- constriction of airway smooth muscle, anticholinergic ing. COPD is associated with a significant level of mor- agents are important as bronchodilators in the treatment bidity and mortality (1, 2). In patients with COPD and of COPD and asthma (3, 4). In clinical trials, anticholin- asthma, bronchoconstriction and mucus secretion are ergics were shown to improve lung function, exercise increased and the airway becomes hyperresponsive to endurance, and health-related quality of life and to reduce exacerbation (5, 6). Treatment with long-acting inhaled *Corresponding author. [email protected] anticholinergic agents improves the effectiveness of Published online in J-STAGE on February 24, 2011 (in advance) pulmonary rehabilitation (7, 8). doi: 10.1254/jphs.10311FP Tiotropium bromide is a synthetic quaternary anticho- 374 Ex Vivo Lung Receptor Binding of Anticholinergics 375 linergic agent approved for maintenance therapy to treat chemicals were purchased from commercial sources. stable COPD, with a once-daily dosage regime providing Anticholinergic agents were dissolved in saline. Each high patient compliance (9, 10). This agent has two im- solution was diluted by 25 mM Tris / 3.75 mM MgCl2 portant properties: it is functionally selective, targeting buffer (pH 7.4) in the in vitro experiment. specific muscarinic receptors that mediate airway smooth muscle contraction, and it has a long duration of action, Animals making it well suited for once-daily dosing (11). It is Male Sprague-Dawley rats (250 – 300 g) at the age of assumed that the mechanism behind its long-lasting ac- 8 – 10 weeks were purchased from Shizuoka Laboratory tion relates to the slow rate of dissociation from its target, Animal Center (Shizuoka). They were housed in the labo- the human M3 muscarinic receptor (12). Ipratropium ratory with free access to food and water and maintained bromide is a short-acting antimuscarinic bronchodilator, on a 12-h light-dark cycle in a room with controlled with a maximal effect at 30 – 60 min and duration of temperature (24 ± 2°C). Animal care and experiments action of 3 – 6 h (13 – 15). Glycopyrrolate bromide is were performed in accordance with the guidelines for the also a well-known quaternary ammonium compound care and use of laboratory animals of the University of endowed with potent antimuscarinic activity. Clinical Shizuoka. studies have shown that inhaled glycopyrrolate displays bronchodilator activity in COPD and asthmatic patients, Intratracheal administration of tiotropium, ipratropium an effect apparently lasting for 8 – 12 h (16 – 18). It was and glycopyrrolate shown that glycopyrrolate inhibited electrical field Rats were fasted for 16 h and then intratracheally (by stimulation–induced contractions of human and guinea- using 1 ml syringe) administered tiotropium (0.6 – 6.4 pig isolated airways with a longer duration of action than nmol/kg), ipratropium (7.3 nmol/kg), and glycopyrrolate ipratropium and that both agents exhibited no selectivity (7.5 nmol/kg) dissolved in saline under temporary anes- toward M1, M2, and M3 receptors (19, 20). thesia with diethyl ether as described previously (23). Because the potency and selectivity of agents for a The intratracheally injected volume of these agents was given receptor in an in vivo setting can be dissimilar to 1.0 ml/kg body weight. The drug diffusion after the in- that observed in an in vitro system, we developed an in tratracheal administration was confirmed by the complete vivo assay to simultaneously determine the absolute po- abolishment of the airway muscle contractile response tency, time course, and organ selectivity of anticholin- induced by the intravenous injection of acetylcholine ergic agents (21). In fact, our previous studies stressed after the tiotropium administration (data not shown) and the importance of characterizing the binding of ligands also by significant occupancy of vasoactive intestinal to receptors under the influence of various pharmacoki- peptide (VIP) receptors in the anterior and posterior lobe netic and pharmacodynamic factors (22 – 27). Although of lungs after the intratracheal administration of the ana- tiotropium, ipratropium, and glycopyrrolate are used logues (23). The control animals received vehicle alone. clinically in inhalable forms to treat COPD and asthma, their binding of muscarinic receptors in vivo has not been Tissue preparation measured in tissues following intratracheal administra- At 2 to 24 h after drug administration, rats were killed tion. Therefore, the aim of the present study was to by taking the blood from the descending aorta under characterize comparatively muscarinic receptor binding temporary anesthesia with diethyl ether. Then the lung, in the lung compared with other tissues of rats treated bladder, and submaxillary gland were dissected and the intratracheally with tiotropium, ipratropium, and gly- fat and blood vessels removed by trimming. The tissues copyrrolate. were minced with scissors and homogenized in a Kine- matica Polytron homogenizer in 19 volumes of ice-cold Materials and Methods 25 mM Tris / 3.75 mM MgCl2 buffer (pH 7.4). The ho- mogenates were twice centrifuged at 40,000 × g for 20 Materials min and the resulting pellets were finally suspended in [N-Methyl-3H]scopolamine methyl chloride ([3H]NMS, buffer for the binding assay. In the ex vivo experiment, 2.59 – 2.65 TBq/mmol) was purchased from PerkinElmer there was a possibility that tiotropium, ipratropium, and Life Sciences, Inc. (Boston, MA, USA). Tiotropium glycopyrrolate might partially dissociate from the recep- bromide (tiotropium) was synthesized by Astellas Co., tor sites during tissue preparation (homogenization and Ltd. (Ibaraki). Ipratropium bromide (ipratropium) and suspension) after drug administration. Yamada et al. (28) glycopyrrolate bromide (glycopyrrolate) were purchased showed that the dissociation of antagonists from recep- from Sigma-Aldrich (St. Louis, MO, USA) and Kempro- tors at 4°C was extremely slow. So, to minimize the tec, Ltd. (Middlesbrough, UK), respectively. All other dissociation, all steps involved in the preparation were 376 M Ogoda et al 3 performed at 4°C. Protein concentrations were measured binding sites (Bmax) for [ H]NMS were estimated. The by the method of Lowry et al. (29). ability of tiotropium, ipratropium, and glycopyrrolate to inhibit [3H]NMS binding (0.5 nM) was estimated from Muscarinic receptor–binding assay the IC50, which is the molar concentration of the anticho- The binding assay for muscarinic receptors was per- linergic agent necessary to displace 50% of the specific formed using [3H]NMS