Journal of Pharmacological and Toxicological Methods
Original articleA novel, quantitative bio-assay for cholecystokinin type-1 receptor activity in the anaesthetised rat
Introduction
Cholecystokinin (CCK) is released from the gastro-intestinal tract in response to a meal and has effects on the secretion and motility of the gastro-intestinal tract by acting as an agonist at CCK1 and CCK2 receptors (Varga et al., 2004, Wank, 1998). While reliable bioassays, readily amenable to pharmacokinetic–pharmacodynamic analysis, are available for compounds acting at CCK2 receptors, CCK1 receptor mediated responses have been more difficult to quantify. Various methods have been tried with varying degrees of success. CCK mediated delay of gastric emptying has been commonly employed as a bioassay for the development of both CCK1 receptor agonists and antagonists in mouse and rat (Bignon et al., 1999, Varga & Scarpignato, 1996, White et al., 2000). However, this method is not very efficient in term of the use of animals and the data obtained tends to be associated with relatively high variance. Collection of the secretion from the bile duct and measurement of pancreatic amylase activity has also been used as a bioassay for CCK1 receptor activation in the rat (Takashi et al., 1997). This method is relatively inefficient as numerous 30 min collection periods are required in addition to the time required for the subsequent determination of the amylase activity of the bile collected. As the plasma concentration of ligands changes over the time course of the assay it is difficult to determine the pharmacokinetic–pharmacodynamic relationship with long duration assays. As a result infusion protocols are adopted that require a relatively large amount of test compound to be administered and in the early stages of a drug discovery program where the supply of such compounds is limited this is burdensome. A further factor that complicates interpretation of CCK1 receptor assays is the fact that the dose–response curve to CCK is commonly found to be bell-shaped (Scarpignato, Kisfalvi, D'Amato, & Varga, 1996). Responses to CCK at CCK1 receptors commonly exhibit tachyphylaxis upon either repeated or continuous exposure. Tachyphylaxis obfuscates the analysis of data obtained in experimental designs based on comparison of the responses to repeated doses within single preparations.
The aim of the current study was to develop an in-vivo model that addressed some of the issues associated with existing models so that responses to CCK and CCK1 receptor antagonists could be rapidly and precisely measured. The rat was the species of choice based on its value for allometric pharmacokinetic scaling and toxicological evaluation. The new assay described is amenable to pharmacokinetic–pharmacodynamic analysis and can be used to assess the effects of CCK agonists and antagonists. Moreover, the techniques developed may be useful for other hormones that stimulate gastro-intestinal smooth muscle contraction.
Section snippets
Surgical procedure
All procedures were performed according to the internationally accepted guidelines for the care and use of laboratory animals in research and were approved by the local IACUC.
Adult Sprague–Dawley rats (300–600 g) were used for all studies. The size of the animal was later found to play a factor in the quality of the preparations. Animals within the weight range 300–400 g were found to provide the most reliable preparations as they had fewer spontaneous contractions of the duodenum relative to
Method development
CCK8S was administered using various intravenous bolus and infusion regimens to find a method that produced a reliable response. Initial studies demonstrated that infusion of CCK8S caused a contractile response of the duodenum but that this response rapidly exhibited tachyphylaxis. The method of intravenous administration was varied such that the rate of infusion, the dose, number of doses and the intervening rest period was varied in an attempt to find a dosing regimen that produced an easily
Discussion
Methods to analyze the CCK1 receptor mediated response, to date, have been difficult to quantify. Most quantitative pharmacological methods assume steady-state conditions (whereas this assay does not make this assumption) and tachyphylaxis is a potentially significant source of error in such bio-assays. The gastric emptying/intestinal transit methods for measuring CCK1 receptor mediated responses are not sufficiently precise and require a relatively large number of animals to obtain meaningful
Acknowledgements
The authors would like to thank Perry Leung in the La Jolla Bio-analytical laboratory of Johnson & Johnson Pharmaceutical Research & Development for determining the plasma concentration of dexloxiglumide.
References (11)
- et al.
Effect of a new CCK-A receptor antagonist, dexloxiglumide, on the exocrine pancreas in the rat
Journal of Physiology (Paris)
(1997) - et al.
Camostate- and caerulein-induced delay of gastric emptying in the rat: Effect of CCK receptor antagonists
European Journal of Pharmacology
(1996) - et al.
Role of endogenous CCK in the inhibition of gastric emptying by peptone and intralipid in rats
Regulatory Peptides
(2000) - et al.
SR146131: A new potent, orally active, and selective nonpeptide cholescystokinin subtype 1 receptor agonist: II. In vivo pharmacological characterization
Journal of Pharmacology and Experimental Therapeutics
(1999) - et al.
Pharmacological study of IQM-97,423, a potent and selective CCK1 receptor antagonist with protective effect in experimental acute pancreatitis
Pharmacology
(2004)
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