SNP's. Next each of these 77 cases was re-examined in the same IWtest-plate to minimise variability. Using this design, 62 no longeryielded IC50 values with non-overlapping CLs. For 7 of the remaining15 cases there were again non-overlapping CLs but this time in theopposite direction. Thus for only 8 compound-SNP combinations werethere IC50s with non-overlapping CLs in the same direction as for thefirst phase (Î″ IC50 values were never N2-fold). In summary, IC50sdefined using the Caucasian WT sequence, at least in this in vitrosystem, are representative of potencies for these relatively commonSNPs.

doi:10.1016/j.vascn.2008.05.081

GLP validation of a hERG patch clamp assay

Nathan Lautermilch, Shimin Wang, Adam Bishop, Teddy Lin, JamesBaumgartner (MDS PHARMA Services, Bothell, WA, USA)

The hERG K+ channel encodes IKr, an important component of thecardiac AP, that contributes to the repolarization of cardiac myocytes.Inhibition of IKr can result in prolongation of the action potential andcause long QT-syndrome, Torsades de Pointes, arrhythmia, andsudden death. Drug induced forms of long QT syndrome have resultedin recent market withdrawals of FDA approved drugs. To identifypotential cardiac liabilities the FDA Guidance for Industry S7BNonclinical Evaluation of the Potential for Delayed VentricularRepolarization specifies that compounds must undergo GLP hERGtesting prior to IND submission. To address these stringent FDArequirements we developed and validated a GLP hERG patch clampassay. During method validation a group of known hERG channelblockers were characterized and results compared with those fromthe literature and values from our PatchXpress automated patch-clamp system. The rank order and absolute potency values generatedbetween the two assays displayed a tight agreement with each otherand reports from the literature. This GLP compliant assay identifiespotential hERG block and allows clients to fulfill regulatory mandatesin a timely and accurate way.

doi:10.1016/j.vascn.2008.05.082

Automated patch clamp screening of hERG and other cardiacchannels using QPatch16

Edward Stevens (NeuroSolutions Ltd., University of Warwick, Coven-try, West Midlands, Cambridge, UK)

Inhibition of the key cardiac ion channels, hERG, TTX-resistantsodium channel and L-type Ca2+ channel can have profound effects onthe duration and amplitude of the cardiac action potential and canresult in arrhythmias and other heart malfunctions. Sophion's QPatchautomated patch technology was used to develop a panel of ionchannel screening assays for predicting cardiac liability of compoundsat an early stage in drug discovery. Both hERG and Nav1.5 assays werebased around stable expression of recombinant channels in CHO celllines, while the L-type calcium channel assay exploited expression ofnative L-type Ca2+ currents in SH-SY-5Y cells. The ion channel assaysare suitable for single point screening or generation of cumulativeconcentration–response relationships. The performance of the hERGQPatch assay was assessed in greater detail. hERG IC50 valuesdetermined from nine compounds spanning 3 orders of magnitudeusing QPatch displayed a good correlation with manual patch clampdata (slope=0.96, R2=0.97, n=3–5). Some automated hERG assaysdisplay marked deviation from manual patch clamp data forhydrophobic ‘sticky’ compounds such as pimozide and terfenadine,

due to compound adsorption to plastic. In contrast, the glass-coatedmicrofluidics of the QPatch reduced compound adsorption giving riseto hERG IC50 values within 3-fold of manual patch clamp data.

doi:10.1016/j.vascn.2008.05.083

Implementation of the 3R's in the rat intravenous drugself-administration model

Joanne Price, D. Hygate, C. Doris, S. Laycock (Aptuit Ltd., ResearchAvenue South, Heriot-Watt Research Park, Riccarton, Edinburgh,Scotland EH14 4AP, UK)

Since the adoption of new guidelines on the non-clinicalinvestigation of the dependence potential of medicinal products bythe EMEA, there has been increased interest in the rodent paradigm ofintravenous drug self-administration (SA). Whilst many studies areperformed using the jugular cannulation model with extrusion of thecatheter at the saddle, from a 3R's perspective this model has a highrisk of blocked catheters that may necessitate re-cannulation ortermination. Using the tail-cuff method of jugular cannulation,previous validation work by Aptuit showed good concordance withpublished data for cocaine; i.e. FR50 achieved in drug substitution test.Animals exhibited very quick recovery from surgery (less invasivethan saddle extrusions), excellent SA response, no catheter blockages,and 100% success rate. Rats maintained for 3months, before sloughingnoted from catheter. Further validation work was carried out using atail-cuff method of femoral vein cannulation. No sloughing wasobserved, no catheter blockages and excellent SA concordance withprevious published data. Animals lasted 4–6 months and remained invery good health— terminated due to out growingmarketed tail-cuffs.Further refinements of the tail-cuff method include the use of amodified cuff made from titanium with strategically placed ventila-tion holes enabling the rats' tail to breathe; and for easy access ifinfection occurs, to replace the industry standard tail-cuff.

doi:10.1016/j.vascn.2008.05.084

Evaluation of memory function in rats: A comparison of watermaze and object recognition tasks

A. Lodiot, P. Rousseau, A.M. Betat, P. Lainee, Forster (CIT, Evreux, France)

Because of the ageing human population and the increasingnumber of drugs prescribed to elderly patients, investigation ofpotential side effects on cognitive function takes increasing impor-tance during safety profile determination of new chemical entities. Inthis study, three designs assessing cognitive abilities either underaversive conditions (CWM: Cincinnati Water Maze; MWM: MorrisWater Maze), or based on the natural exploratory behaviour of rats(ORT: Object Recognition Task) were tested using the same pharma-cological agent, scopolamine (SCO) at 3 mg/kg ip. In the ORT, SCOreduced the performance at 2 h by 90%. The learned improvement inescape time during trials conducted from day 1 to day 4 in the MWMwas reduced by SCO treatment (60% vs. 80% in controls on Day 4). Inthe CWM, SCO induced no significant modifications during thelearning and memorization phases, but adaptation to environmentalchanges was reduced (2 to 4 fold increase of failure rate).It is wellknown that the cholinergic system is involved in the memory processand SCO is commonly used to induce amnesia. However, othermechanisms (glutamatergic regulation) are also involved in memoryprocess, requiring other reference items to be tested. As a simple, rapidand reliable test, ORT could be used as a primary test in the evaluationof memory. The use of mazes enables the investigation of the multiple

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