Sonia P. Roberts

Analysis of variation in L-365,260 competition curves in radioligand binding assays.

1 . For several years, we have used the cholecystokinin (CCK)B/gastrin receptor selective antagonist, L‐365,260, as a reference compound in a variety of studies in CCKB/gastrin receptor radioligand binding assays. Here, we have analysed the competition curve data sets obtained between L‐365,260 and [125I]‐BH‐CCK8S in guinea‐pig gastric gland and mouse and rat cerebral cortex preparations. 2 . Competition curves obtained for L‐365,260 in the mouse cortex assay were not different from rectangular hyperbolae (slope = 1.01 ± 0.02) implying the presence of a single population of binding sites (pKI = 8.41 ± 0.01; data from 47 experiments, slope constrained to unity). However, in the rat cortex and guinea‐pig gastric gland assays, the mean slope of the competition curves was significantly less than one and the mean apparent pKI significantly lower than that obtained in the mouse cortex (slope = 0.85 ± 0.03, 0.90 ± 0.03; apparent pKI = 7.98 ± 0.05, 8.07 ± 0.05; 48 and 45 experiments, in rat and guinea‐pig, respectively). The distribution of the individual pK1 and slope estimates of the competition curves in these two assays was consistent with expectations for the variable expression (in terms of absolute number and proportion) of two binding sites. The two sites were characterized by pK1 values for L‐365,260 of 8.50 ± 0.04 and 8.48 ± 0.04 for the high affinity site and 7.32 ± 0.04 and 7.22 ± 0.06 for the low affinity site in guinea‐pig and rat, respectively. 3 . The affinity estimates for L‐365,260, although obtained on different tissues, are consistent with data obtained from the analysis of L‐365,260 antagonism of pentagastrin‐stimulated responses in mouse and rat stomach (acid secretion) and guinea‐pig gastric muscle (isotonic contraction) assays. To this extent, these data suggest the existence of two CCKB/gastrin receptor subtypes.

Pharmacological analysis of the CCKB/gastrin receptors mediating pentagastrin-stimulated gastric acid secretion in the isolated stomach of the immature rat

1 The CCKB/gastrin receptors mediating pentagastrin stimulation of gastric acid secretion by histamine release and by direct stimulation of oxyntic cells have been characterized in the immature rat isolated stomach assay. This was achieved by estimating antagonist affinity values for competitive antagonists from three distinct chemical classes (L‐365,260, PD 134,308 and JB93190) in the absence and presence of a high concentration of the histamine H2‐receptor antagonist, famotidine (30 μm). 2 Pentagastrin produced concentration‐dependent stimulation of gastric acid secretion in the absence and presence of famotidine. Famotidine depressed the maximum secretory response to pentagastrin although the degree of depression varied between experimental replicates (25–60%). This variation was attributed to the histamine‐release mediated component of acid secretion, as judged by the consistency of the maximum responses obtained in the presence, but not absence, of famotidine. 3 All three CCKB/gastrin receptor antagonists behaved as surmountable antagonists in the absence and presence of famotidine. JB93190 (pK∼B∼9.1, ∼8.9, in the absence and presence of famotidine, respectively) was approximately 30 fold more potent than either L‐365,260 (pKB∼7.4, ∼7.1) or PD 134,308 (pKB∼7.6, ∼7.4). 4 It was assumed that the famotidine treatment converted pentagastrin‐stimulated acid secretion from a combination of an indirect action due to the release of histamine and a direct action on the oxyntic cell to solely a direct action on the oxyntic cell. A simple mathematical model of this two‐receptor system was developed. The direct and indirect components were assumed to sum to produce the total response to pentagastrin obtained in the absence of famotidine. It was found that this model could account quantitatively for the behaviour of the three antagonists without invoking a difference in antagonist affinity for the CCKB/gastrin receptors mediating the direct and indirect actions of pentagastrin. However, a conclusion of receptor homogeneity has to be qualified because the model was also used to generate simulations which indicated that the analysis could only detect antagonist affinity differences of greater than one log‐unit between enterochromaffin‐like (ECL) and oxyntic cell CCKB/gastrin receptor populations.

Achiral, selective CCK2 receptor antagonists based on a 1,3,5-benzotriazepine-2,4-dione template.

Novel, achiral 1H-1,3,5-benzotriazepine-2,4(3H,5H)-diones have been prepared and structurally characterized. These compounds are potent CCK(2) receptor antagonists that display a high degree of selectivity over CCK(1) receptors.

Analysis of the variation in the action of L‐365,260 at CCKB/gastrin receptors in rat, guinea‐pig and mouse isolated gastric tissue assays

1 . Since L‐365,260 was first described as a selective antagonist at cholecystokinin (CCK)B/gastrin receptors, we have used it periodically as a reference compound in isolated tissue assays of guinea‐pig gastric muscle and lumen‐perfused stomachs from mouse and immature rat. L‐365,260 behaved as a surmountable antagonist and produced parallel rightward shifts of pentagastrin con***ćentration‐effect curves in each of the replicate experiments. The experiments were performed by several different experimenters in the same laboratories over a five year period. 2 . In the isolated, lumen‐perfused, immature rat stomach assay, L‐365,260 behaved as a simple competitive antagonist (Schild plot slope = 1.00 ± 0.10, pKB = 7.54 ± 0.03 from a global analysis of the data) acting at a homogeneous population of receptors in five separate, highly‐reproducible, experiments. In contrast, the replicate data sets obtained from the interaction in the isolated, lumen‐perfused mouse stomach and guinea‐pig gastric muscle assays, over the same period, were not consistent with the presence of a single receptor population. The guinea‐pig gastric muscle data were relatively reproducible between experiments but some individual Schild plot slopes and the slope estimated from a global analysis of all the data were significantly less than unity (slope = 0.80 ± 0.07, pA2 = 8.56 ± 0.05 from the global analysis). The data obtained in the mouse stomach were significantly more variable than that obtained in the same assay, during the same period, from the interaction between histamine and the H2‐receptor antagonist, famotidine. The individual Schild plot slopes ranged from being very flat (0.20) to being not significantly different from unity (1.23) and the pA2 values ranged from 7.68 to 8.70. 3 . Overall, the data could be accounted for by assuming the variable expression of two receptor subtypes across the assays. The rat stomach appeared to express a single receptor characterized by a low affinity constant for L‐365,260 (pKB ∼ 7.5). The guinea‐pig gastric muscle and mouse stomach data could be explained by the presence of this receptor and a second one characterized by a high affinity constant for L‐365,260 (pKB ∼ 8.6). The activity of the two proposed receptor subtypes was consistent between experiments in the guinea‐pig and the high affinity receptor appeared to be predominant. In contrast, the mouse stomach data could only be simulated by assuming that the proportion and absolute number of each subtype varied significantly between the replicate experiments. 4 . The L‐365,260 affinity estimates at the inferred receptor subtypes were indistinguishable from those obtained in a corresponding analysis of the behaviour of L‐365,260 in CCKB/gastrin receptor radioligand binding experiments in guinea‐pig gastric gland and mouse and rat cerebral cortex preparations.

Thermodynamic analysis of ligands at cholecystokinin CCK2 receptors in rat cerebral cortex

Several studies using radioligand binding assays, have shown that measurement of thermodynamic parameters can allow discrimination of agonists and antagonists ( Weiland et al., 1979 ; Borea et al., 1996a ). Here we investigate whether agonists and antagonists can be thermodynamically discriminated at CCK2 receptors in rat cerebral cortex.

Optimization of Platelet-Derived Growth Factor Receptor (PDGFR) Inhibitors for Duration of Action, as an Inhaled Therapy for Lung Remodeling in Pulmonary Arterial Hypertension.

A series of potent PDGFR inhibitors has been identified. The series was optimized for duration of action in the lung. A novel kinase occupancy assay was used to directly measure target occupancy after i.t. dosing. Compound 25 shows 24 h occupancy of the PDGFR kinase domain, after a single i.t. dose and has efficacy at 0.03 mg/kg, in the rat moncrotaline model of pulmonary arterial hypertension. Examination of PK/PD data from the optimization effort has revealed in vitro:in vivo correlations which link duration of action in vivo with low permeability and high basicity and demonstrate that nonspecific binding to lung tissue increases with lipophilicity.

Inhibitors of Gastric Acid Secretion

The discovery of potent and selective inhibitors of gastric acid secretion now negates the need for surgical intervention in the treatment of many hypersecretory disorders. The side effects associated with cholinergic antagonists has led to their being superseded by the use of selective histamine H2-receptor antagonists (cimetidine, ranitidine, famotidine) and the more potent irreversible proton-pump inhibitors (omeprazole, rabeprazole, lansoprazole, pantoprazole, esomeprazole). Current therapeutic regimes for the treatment of ulcer disease recommend concurrent proton-pump administration while eradicating Helicobacter pylori, a bacterial infection that is strongly associated with ulcer formation. Because achlorhydria increases the likelihood of gastrointestinal infection, current research within this area is now targeting the design of reversible proton-pump inhibitors and CCK2/gastrin-receptor antagonists. These newer therapeutic approaches, however, are unlikely to produce the rapid and potent inhibition of acid secretion realized by the irreverisble PPIs, which in addition have few side effects and a proven track record of success. Key drug developments in the treatment of acid hypersecretion, from the anticholinergic agents to the highly potent and effective proton-pump inhibitors currently in use, are covered in this chapter. Keywords: gastric acid; ulcer; histamine; acetylcholine; CCK2/gastrin; proton pump; H2-receptor antagonist; proton-pump inhibitor