Preclinical evaluation of long-acting muscarinic antagonists: comparison of tiotropium and investigational drugs.

Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation caused by persistent inflammatory processes in the airways. An increased cholinergic tone mediates different pathophysiological features of COPD, such as bronchoconstriction and mucus hypersecretion, mostly through activation of the human muscarinic M(3) receptor (hM(3)) subtype. Tiotropium bromide (Spiriva) is a well established muscarinic antagonist in the pharmacological management of COPD with a once-daily posology. The rationale behind the sustained bronchodilation obtained with tiotropium consists in its slow dissociation from hM(3) receptors. In this study, we performed a comprehensive preclinical comparison of tiotropium with other long-acting muscarinic antagonists (LAMAs) currently in clinical development, namely aclidinium bromide and glycopyrrolate. The different muscarinic antagonists were characterized for their 1) affinity toward the different human muscarinic receptor subtypes expressed in Chinese hamster ovary cells and kinetics of receptor dissociation, 2) potency in inhibiting the agonist-induced activation of muscarinic receptors through measurement of second messengers, and 3) efficacy and duration of bronchoprotection, as tested in a model of acetylcholine-induced bronchoconstriction in anesthetized dogs over a period of 24 h. All of the tested LAMAs showed high affinity and potency toward the hM(3) receptor (tiotropium, pA(2) = 10.4; aclidinium, pA(2) = 9.6; and glycopyrrolate, pA(2) = 9.7). However, dissociation half-lives of the LAMAs from the hM(3) receptor differed significantly (tiotropium, t((1/2)) = 27 h; aclidinium, t((1/2)) = 10.7 h; and glycopyrrolate, t((1/2)) = 6.1 h). In line with their kinetic properties at the hM(3), the tested LAMAs provided different levels of bronchoprotection in the in vivo setting 24 h after administration (tiotropium = 35%, aclidinium = 21%, and glycopyrrolate = 0% at 24 h) when applied at equieffective doses.

New dopamine D2 receptor polymorphisms in rats and association with apomorphine-induced stereotypies.

Adult Wistar rats injected with the dopamine receptor agonist apomorphine display different types of motility patterns with respect to oral stereotypes and locomotor activities. It was tested whether phenotypes exhibiting either 'sniffing' or 'non-sniffing' behaviour differed in gene structures of dopamine receptors D1 or D2. Forty-five Wistar rats of both genders were tested after a single dose of apomorphine (2 mg/kg s.c.) for stereotyped behaviour. Sequence analysis of the 5' flanking region, the 5' untranslated region and the coding region of the two genes revealed a new sequence for the 5' flanking region of the D1 receptor gene and two polymorphisms in the promoter region of the D2 receptor gene. The D2 receptor polymorphisms were denominated as T-355C- and A-257G-polymorphism. Between 'sniffing' and 'non-sniffing' animals no differences were detected in their D1 receptor sequence but there was a significant (P<0.05) association of the two phenotype groups with the D2 receptor genotype. These findings indicate that in Wistar rats at least part of an individual apomorphine stimulated behavioural phenotype might be due to an individual dopamine receptor D2 genotype.

Developing a strategy for the nonclinical assessment of proarrhythmic risk of pharmaceuticals due to prolonged ventricular repolarization.

The aspects for developing a strategy for the preclinical testing of drug candidates for proarrhythmic potential are presented. The rationale for such a strategy reflects primarily the needs for efficient and scientifically based drug development and also attempts to anticipate the possible outcomes of the currently ongoing regulatory activity (ICH S7b and E14). Whereas a wealth of new data have emerged over the past few years, demonstrating the utility of test systems for detecting drug effects on myocardial repolarization, the current regulatory trend appears to not use such data for the clinical trial design or risk assessment. Nevertheless, certain types of preclinical tests are highly recommended for optimizing drug development, despite their still questionable regulatory acceptance. This includes (1) testing for blockade of I(Kr) or hERG-mediated potassium current in heterologous cell systems, (2) measurement of effects on the myocardial action potential in vitro; and (3) assessment of effects on the ECG in a well-conducted in vivo study. Due to their requirement for little compound, the first two in vitro tests lend themselves for early safety testing of drug candidates still in the lead optimization phase of drug discovery; together, they form a useful and predictive in vitro assessment. This strategy is not new but reflects what was initially suggested by the Committee for Proprietary Medicinal Products (CPMP) some years ago. However, the validation of such a strategy and its utility in drug development is now well established and recommended, independent from future regulatory requirements.