Optimisation and validation of a medium-throughput electrophysiology-based hERG assay using IonWorks HT.

INTRODUCTION: Regulatory and competitive pressure to reduce the QT interval prolongation risk of potential new drugs has led to focus on methods to test for inhibition of the human ether-a-go-go-related gene (hERG)-encoded K+ channel, the primary molecular target underlying this safety issue. Here we describe the validation of a method that combines medium-throughput with direct assessment of channel function. METHODS: The electrophysiological and pharmacological properties of hERG were compared using two methods: conventional, low-throughput electrophysiology and planar-array-based, medium-throughput electrophysiology (IonWorks HT). A pharmacological comparison was also made between IonWorks HT and an indirect assay (Rb+ efflux). RESULTS: Basic electrophysiological properties of hERG were similar whether recorded conventionally (HEK cells) or using IonWorks HT (CHO cells): for example, tail current V1/2 -12.1+/-5.0 mV (32) for conventional and -9.5+/-6.0 mV (46) for IonWorks HT (mean+/-S.D. (n)). A key finding was that as the number of cells per well was increased in IonWorks HT, the potency reported for a given compound decreased. Using the lowest possible cell concentration (250,000 cells/ml) and 89 compounds spanning a broad potency range, the pIC50 values from IonWorks HT (CHO-hERG) were found to correlate well with those obtained using conventional methodology (HEK-hERG)(r=0.90; p<0.001). Further validation using CHO-hERG cells with both methods confirmed the correlation (r=0.94; p<0.001). In contrast, a comparison of IonWorks HT and Rb+ efflux data with 649 compounds using CHO-hERG cells showed that the indirect assay consistently reported compounds as being, on average, 6-fold less potent, though the differences varied depending on chemical series. DISCUSSION: The main finding of this work is that providing a relatively low cell concentration is used in IonWorks HT, the potency information generated correlates well with that determined using conventional electrophysiology. The effect on potency of increasing cell concentration may relate to a reduced free concentration of test compound owing to partitioning into cell membranes. In summary, the IonWorks HT hERG assay can generate pIC50 values based on a direct assessment of channel function in a timeframe short enough to influence chemical design.

Differential sensitivity of antinociceptive assays to the bradykinin antagonist Hoe 140.

1. The antinociceptive activity of the bradykinin (BK) BK2 receptor antagonist D-Arg-[Hyp3,Thi5D-Tic7,Oic8]BK (Hoe 140) was determined in a range of mouse abdominal constriction assays. 2. Hoe 140 potently inhibited the response induced by i.p. injection of 10 micrograms BK/mouse, and 1 microgram BK/mouse in mice pre-sensitized by i.p. injection of prostaglandin E2 (PGE2). The ED50 values in these assays were 1.9 and 3.7 micrograms kg-1 respectively. This confirms that Hoe 140 is a potent antagonist of BK in vivo. 3. Hoe 140 produced potent, but incomplete inhibition of the responses evoked by i.p. injection of kaolin or 0.25% acetic acid. ED25 values in these assays were 2.7 and 16.1 micrograms kg-1, and the maximum inhibition produced was 60% and 70% respectively. 4. At doses up to 1 mg kg-1, Hoe 140 was completely ineffective against the abdominal constriction response induced by zymosan. In contrast, morphine, ibuprofen and indomethacin had similar potencies against zymosan, kaolin and acetic acid-induced abdominal constriction. 5. Although zymosan, acetic acid and kaolin all produce qualitatively similar responses, it is appears that they achieve this by different mechanisms. The extent to which BK is involved as a mediator differs between the various types of abdominal constriction assay.

Unilateral 5,7-dihydroxytryptamine lesions of the dorsal raphe nucleus (DRN) and rat rotational behaviour.

A unilateral lesion in the dorsal raphe nucleus (DRN) resulted in a decreased concentration of 5-hydroxytryptamine (5-HT) in the ipsilateral striatum (CS), anterior cortex and substantia nigra (SN), a loss of [3H]5-HT uptake sites in the cortex and striatum and a selective reduction in 5-HT turnover in the substantia nigra. The directly acting 5-HT agonist 5-methoxy-N,N-dimethyltryptamine induced contralateral turning behaviour in the lesioned animals, whilst the 5-HT releasing agent, p-chloroamphetamine, induced ipsilateral rotation. All rotational behaviour was blocked by haloperidol and the turning behaviour in response to 5-MeODMT was blocked by methysergide. The data presented suggest that the DRN innervates the SN and CS differentially and that nigral 5-HT receptors become supersensitive after denervation of the DRN-SN pathway.

Supersensitivity of nigral serotonin receptors and rat rotational behaviour.

Direct injections of 5-hydroxytryptamine (5-HT) into the corpus striatum (CS) and substantia nigra (SN) after unilateral 5,7-dihydroxytryptamine (5,7-DHT) of the dorsal raphe nucleus (DRN), resulted in a significant dose related contralateral turning behaviour in SN-injected but not in CS-injected animals. Receptor binding studies in these animals revealed a 3 fold increase in [3H]5-HT binding sites (Bmax) and a similar increase in affinity constant (KD) in the SN from the lesioned side when compared with the unlesioned side. There was no change in binding characteristics in the CS of DRN-lesioned animals. The data presented would confirm our previous findings that nigral 5-HT receptors become supersensitive after denervation of the DRN-SN pathway.

Possible mechanism of 5-methoxy-N,N-dimethyltryptamine-induced turning behaviour in DRN lesioned rats.

5-Methoxy-N,N-dimethyltryptamine (5-MeODMT) (7.5 mg/kg SC) caused a contralateral turning in rats with a unilateral lesion of the dorsal raphe nucleus (DRN). This turning behaviour was blocked by pretreatment with putative 5-HT antagonists, methysergide, cyproheptadine and cinanserin. The peripheral 5-HT antagonist, xylamidine, also prevented the response to 5-MeODMT. Of the other neurotransmitter antagonists, only haloperidol was active, hyoscine, picrotoxin, naloxone and strychnine were ineffective. Pretreatment with alpha-methyl-p-tyrosine (alpha-MT) also significantly reduced the turning response to 5-MeODMT. These results indicate that a central dopaminergic system is involved in 5-MeODMT-induced turning behaviour. This suggestion is supported by the finding that an ipsilateral turning in response to 5-MeODMT was observed in the rats with additional 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle (MFB). The possible mechanisms by which 5-MeODMT induced turning in DRN lesioned rats are discussed.

Effects of Zeneca ZD7288 in comparison with alinidine and UL-FS 49 on guinea pig sinoatrial node and ventricular action potentials.

ZENECA ZD7288 (4-(N-ethyl-N-phenyl-amino)-1,2-dimethyl-6-(methylamino) pyrimidium chloride) is a novel compound which we compared with alinidine and UL-FS 49 (zatebradine) in guinea pig sinoatrial node (SAN) and papillary muscle preparations, using conventional microelectrode techniques. At low concentrations (1 x 10(-8)-1 x 10(-6) M), ZD7288 caused slowing of the diastolic depolarisation rate of SAN pacemaker cells, thus prolonging the diastolic interval and slowing the beating rate. Alinidine and UL-FS 49 also had qualitatively similar effects on diastolic depolarisation rate, but ZD7288 caused least prolongation of the action potential duration (APD) of SAN cells at the concentrations that had "selective bradycardic actions." ZD7288 affected the APs of ventricular cells in guinea pig papillary muscle only at relatively high concentrations (3 x 10(-6)M-1 x 10(-4) M), which reduced plateau potential duration, although total APD was less affected. Reduced force of contraction (FOC) was also observed at these high concentrations; significant effects on AP Vmax were noted only at concentrations > or = 3 x 10(-5)M. Alinidine also had negative inotropic effects on papillary muscle, but its effects were noted at concentrations similar to those with bradycardic actions; in contrast, UL-FS 49 had marked positive inotropic actions and also increased ventricular APD within the bradycardic concentration range. These data provide a basis for the selective actions of ZD7288 on heart rate (HR).

Effects of small-particle aerosols of local anaesthetic on dyspnoea in patients with respiratory disease.

This study was devised to test the hypothesis that dyspnoea could be mediated by unmyelinated vagal sensory nerve endings (type J receptors) situated at alveolar level in the lung. A modified jet nebulizer was used to generate an aerosol of local anaesthetic in particles small enough to allow alveolar deposition. Lignocaine (2% and 5%) produced aerosols with an arithmetic mean diameter (+/- SD) of 1.5 +/- 0.3 and 1.2 +/- 0.6 micron respectively, the mass median diameters being 1.7 (geometric standard deviation = 1.2) and 2.5 (geometric standard deviation = 1.7) micron respectively. In experimental animal models a vagally mediated tachypnoea may be induced acutely by pulmonary microembolism. This response is known to be mediated by unmyelinated pulmonary afferent nerves in the vagus. Local anaesthetic agents administered as small particles, but not as large particles, obtunded this response, which suggests that the aerosol was capable of penetration to alveolar level. Upon this background, a clinical study was designed to compare the effects of lignocaine with placebo both given as small-particle aerosols. Six patients, including two with diffuse alveolar pathology and four with chronic airflow obstruction, were studied. Respiratory frequency was determined before and after the aerosol, and exercise tolerance and breathlessness were measured with a 6 min walking test and visual analogue scales. After lignocaine there was no clinical evidence of anaesthesia of the upper airways but bronchoconstriction occurred. While no overall effect of lignocaine on dyspnoea was apparent, individual patients showed some evidence of benefit.(ABSTRACT TRUNCATED AT 250 WORDS)