Comparison of dogmyocytes and purkinje fibres for assessment ofdrug-induced changes in action potential duration

Najah Abi-Gerges, Jean-Pierre Valentin, Chris E. Pollard (SafetyAssessment UK, AstraZeneca R&D Alderley Park, Macclesfield, UK)

Dog Purkinje fibres (PF) are often used to test the effect of drugs onaction potential duration (APD), but throughput is low and animal usagehigh.We investigated dogmid-myocardial (M) cells as an alternative. APsin dog PFs or M-cells were recorded using the same sharp-electrodemethodology to measure APD90 at either 1 or 0.5 Hz. Data are mean±SEM (n=3–8 cells/fibres; ⁎pb0.05 vs values inM-cells). Avalidation set of6 standards was used (cisapride, diltiazem, dofetilide, pinacidil, D-sotalol,terfenadine). For each, concentration-effect curve data were comparedto vehicle (0.1% DMSO). Representative data for 3 compounds areshown. D-sotalol increased APD90 at 1 Hz (at 100 µM28±3% and 58±10%inM-cells andPFs, respectively). This effectwas reverse use-dependent inboth preparations (at 0.5 Hz, 37±6% and 77±17% in M-cells and PFs,respectively). In M-cells, terfenadine had a biphasic effect on APD90 at 1and 0.5 Hz, consistent with inhibition of multiple ion channels. At 1 Hz itcauseda small increase inAPD90at0.1µM(6±2%)but at 10µMdecreasedAPD90 (−74±2%). It had no effect on APD90 in PFs. At both frequencies,3 µM pinacidil decreased APD90 to a greater extent in PFs (at 1 Hz: −41±5%⁎ vs −9±2%). However, the effect in M-cells was similar to that in PF at10 µM (−51±3%). M-cells may be a suitable alternative to PF: throughputis 4×higher, animal usage is reduced4-fold, there is nodiffusionbarrier tolimit drug access and, since M-cell action potential repolarisation isreported to define the end of the T-wave, M-cell datamay better relate toQT measurements in dogs.

doi:10.1016/j.vascn.2008.05.074

Applicarion of isolated heart electrophysiology model forinvestigative studies of cardiac toxicity

Danshi Li, Jia Zhu, Hong Shi, Lucy Sun, Paul Levesque (Bristol-MyersSquibb, Pennington, NJ, USA)

The isolatedheart EPmodel is used topredictQT interval prolongationand associated proarrhythmia, however, this model is also useful formechanistic studies of other CV liabilities. The mechanistic basis of CVtoxicities of two late discovery leads was evaluated in isolated heartsinstrumentedwith ECG electrodes, a coronary artery flow (CF) probe, andatrial pacing electrodes for assessing sinoatrial (SA) node function (HRand SA node recovery time or SNRT), and atrioventricular (AV) nodefunction. A clinical development candidate produced bradycardia andsinus arrest in dog TK studies and themechanismwas evaluated in rabbithearts. The compound decreased sinus rate and increased SNRT,indicating a direct inhibitory effect on SA node function. The effectswere closely mimicked by the pacemaker channel inhibitor zatebradine,and couldbe reversedby isoproterenol. SARgenerated in themodel led tothe identification of potent analogs devoid of SA node activity. A clinicalcandidate in another program produced cardiac lesions potentiallyassociated with vasoconstriction in rat tox studies. CV channel andreceptor profiling assays were negative, except for activity in anadenosine A2a radioligand binding assay. A2a active analogs reduced CFin isolated rat hearts, similar to the A2a antagonist ZM-241385, and theselective A2a agonist CGS-21680 abolished the CF effect. An A2a inactiveanalog did not reduce CF and or produce lesions in rats. The studiesdemonstrate the utility of the isolated heart EP model in mechanisticevaluation of diverse cardiac toxicity.

doi:10.1016/j.vascn.2008.05.075

Effects of JNJ-17333030, naratriptan and sumatriptan on humanisolated coronary artery

SandraWilliamsa, Bob Sheldricka, David Gallacherb, Thomas Stecklerb,Rob Towartb (AsterandUKLtd., Royston,Herts, UK) ( Johnson& JohnsonPharmaceutical Research & Development, a Division of JanssenPharmaceutica, Turnhoutseweg 30, Beerse, Belgium)

Coronary constriction is a known side effect of some drugs such astriptans. In the current study, the coronary constrictor potential of JNJ-17333030, a 5HT reuptake inhibitor/alpha 2 antagonist, was comparedto naratriptan and sumatriptan in human isolated coronary artery(HCA) as part of its safety evaluation. HCA rings from bothatherosclerotic (as) and non atherosclerotic (nas) vessels, two donorsof each, were suspended in tissue baths for measurement of musclecontraction. KCl (30–100 mM) and PGF2a (1 mM) caused contractions,whereas sodium nitroprusside (100 mM) caused relaxation in all HCArings examined (16 nas, 12 as). Substance P (1 nM) caused relaxationin the majority of HCA rings (15 nas and 8 as). The magnitude of theseresponses was significantly greater in nasHCA compared to asHCA (P£0.01). JNJ-17333030 (0.1 nM–1 mM) did not cause contraction (n=8rings from 4 donors), whereas naratriptan and sumatriptan causedconcentration-dependent contractions in both nas and asHCA.Naratriptanwas approximately 10-fold more potent than sumatriptanin nasHCA (pEC50 7.2±0.1 and 6.2±0.1, respectively, n=4 rings from 2donors) but was equipotent in asHCA (pEC50 6.8±0.1 and 6.7±0.1,respectively, n=3 rings from 2 donors); contractions were similar inmagnitude but were larger in nasHCA. In conclusion, JNJ-17333030 didnot contract HCA from either vessel type, whereas both naratriptanand sumatriptan did. The magnitude of the responses to allcompounds examined was greater in nasHCA, demonstrating thatasHCA are less responsive, possibly as a consequence of arterialstiffness.

doi:10.1016/j.vascn.2008.05.076

Characterisation of an ionworks-based assay for thehKCNQ1/hKCNE1 (IKs) cardiac ion channel

M.H. Bridgland-Taylor, C.M. Gorvin, J.M. Ellston, J.-P. Valentin, M.Main, C.E. Pollard (Safety Assessment & Biological Chemistry,AstraZeneca R&D Alderley Park, Macclesfield, UK)

Although the hERG channel is well established as the primarymechanism for drug-induced QT prolongation, there is good geneticevidence (LQTS1 and 5) that inhibition of the channel complexcarrying IKs (hKCNQ1/hKCNE1) is an alternative mechanism. Further-more, drug-like molecules have been shown to inhibit this channeltype (e.g. indapamide, linopirdine). We sought to characterise an IKsassay using a commercially-available CHO cell line (Millipore). All dataare mean ± sem, except for potency data: mean; lower, upper 95%confidence limit (n was 6–9, except where stated). In conventionalwhole-cell electrophysiology the current showed characteristicsreported in other IKs cell lines (e.g. Dong et al., 2006 (J Mem Biol210, 183–192). Specifically, activation V1/2 31.1±0.5 mV; slope factor(k) 18.1±0.5. Activation rate was slow in absolute terms but voltagedependent (e.g. 8.55±1.95 s at +30 mV; 2.60±0.56 s at +80 mV), aswas deactivation rate (e.g. 1.10±0.15 s at −30 mV; 0.31±0.04 s at−120 mV). The IKs blocker XE-991 (10 uM) gave 85.0±4.1% inhibitionand an activator of IKs (AZ11880459 (AZ2)) shifted the voltage-dependence of activation (V1/2: control 32.7±1.9mV; 30 uMAZ2 22.0±1.0 mV; Pb0.05; Student's t-test). Data were qualitatively similar inIonWorks: V1/2 29.0±1.1 mV; k 12.0±0.6; XE-991 IC50 0.38; 0.35,0.41 uM (n=26–39); 30 uMAZ2 increased current amplitude at +40mVby 44.1 ±4.8%. In summary, preliminary IonWorks data for the

164 Journal of Pharmacological and Toxicological Methods 58 (2008) 147–178