Cell Lines-cytobioscience

1. BIOSCIENCE CRO Services www.cytobioscience.com 1

2. CRO Services Why partner with us? OuR SpeCialty iS in vitro and in vivo Safety phaRmaCOlOgy We dOn’t juSt • ContractedbytheFDAtoprovideionchannelscreening servicesinsupportoftheCiPAintiative • Recognizedexpertsandthoughtleaders • Clientbasedstudydesignandcontextorientedresults pROvide data, We pROvide anSWeRS WIth DeCADes oF IoN ChANNel AND IN vIvo exPerIeNCe, CytOBiOSCienCe iS uniquely pOSitiOned tO Be yOuR One StOp ShOp fOR Safety phaRmaCOlOgy BIOSCIENCE FromtheCiPAionchannelpaneltostemcellstoin vivostudies,wecarry outallaspectsofthesafetycharacterizationofyourcompounds.We alsoofferGLPcapabilitiesanddoseformulationanalysis. We offer higher throughput ion channel screening services using our CytoPatch – the „hands-free“ manual patch clamp platform. With our portfolio of capabilities, we can support screening of compounds from discovery through IND enabling studies. We are proud to have been contracted by the FDA to provide ion channel screening services in support of the CiPA intiative. references for ion channel screening service using CytoPatch: ASSAY and Drug Development Technologies 2014 Oct. Vol. 12 No.8: 457-469 Action Potential Characterization of Human Induced Pluriopotent Stem Cell-Derived Cardiomyocytes Using Automated Patch-Clamp Technology | Scheel O, Frech S, Amuzescu B, Eisfeld J, Lin K-H, Knott T F1000 Research 3:245 · October 2014 Late cardiac sodium current can be assessed using automated patch-clamp | Chevalier M, Amuzescu B, Gawali V, Todt H, Knott T, Scheel O, Abriel H Drug Discovery World Spring 2013: 60-66 An automated approach to solving Pharma‘s cardiac toxicity conundrum | Van de Waart B, Westerink W, Pineiro Costas N 2

3. We have pROven expeRienCe in CaRdiOvaSCulaR ReSeaRCh SinCe 1996 eleCtRiCal aCtivity Of the heaRt the electrical activity of the heart ismanifestedatthewholeorganle- velasthewaveformknownastheelectrocardiogram(ECG).Thisisthe resultofelectricalimpulsesgeneratedinthesino-atrialnode(SAN)con- ductingthroughouttheatria,throughtheatrio-ventricularnode(AVN) andthenintotheventricleviatheHis-Purkinjesystem.Underlyingthe overallelectricalactivityoftheheartareactionpotentialsgeneratedfrom eachoftheelectricallyexcitablecellsintheheart.Furtherdissectionof theelectricalactivityoftheheartrevealsthatavarietyofionchannels areresponsibleforgeneratingeachactionpotential.Theseionchannels carrycurrentintheformofionswhichmoveintoandoutofthecell. Overthepastdecades,manystudieshaveshownthatblockadeofthe- secardiacionchannelscanleadtoavarietyofarrhythmias.Therefore, themostdirectmethodofevaluatingthecardiacsafetyofacompound istomeasureitseffectsoncardiacionchannels. the cardiac action potential (AP).TheAPisclassicallydividedintopha- ses(0-4).SurroundingthisAParetheioncurrentswhicharerespon- sibleforthedifferentphases.Ioncurrentswhichareoutward(upward direction)repolarizethecellorreturnittoarestedstate,whereas,ion currentswhichareinward(downwardindirection)depolarizethecellor excitethecell. Illustration of a transected ion channel in a cell membrane.Ionchannels aremembraneboundproteinswhichallowthepassageofionsintoand outofthecell.Blockadeoftheionchannelbyadrugcaninterruptthepas- sageoftheseionsandcauseelectricaldisturbancesintheheart. 3

4. CaRdiaC iOn Channel aSSayS paCkageS: Individualionchannelevaluations CiPAionchannelpanel Chronicexposure/channeltrafficking CharacterizationofthemechanismofQRS,PR,QTintervalprolongation stuDy: Manualorautomatedpatchclamp GLPornon-GLP Recordingconditions: Asclosetophysiologicaspossible(temperature,voltagewaveforms,ionconcentrations) Cell tyPe: Expressionsystems acutelyisolatedhumanatrialorventricularmyocytes iPS-cardiomyocytes turNArouND: 2-5daysfromreceipttodraftreport(dependingonassaytypeandcompoundnumber) More information on human cardiac ion channels see: Crumb WJ, Jr., et al. Mol Pharm 1995; 47:181-190.; Crumb WJ, Jr, et al. Am J Physiol 1995; 268:H1335-H1342.; Crumb WJ, Jr, et al. Circ Res 1995; 77:950-956.; Roca TP, et al. Pedi- atric Res 1996; 40:462-468.; Crumb WJ, et al. JPET 1999; 289:386-391.; Crumb WJ, Jr. British J Pharmacol 1999; 126:575-580.; Crumb WJ Jr., Cavero I. Current Protocols in Pharmacol 2002; 10.8.1-10.8.17 4

5. We explain data and put ReSultS in COntext fOR OuR CuStOmeRS. iOn Channel BlOCk Can lead tO diffeRent aRRhythmiaS Block of potassium currents canleadtoQTprolongation andtorsadedepointes Block of sodium current (ina) canleadtoventricular tachycardia Block of calcium current (ica) canleadtoAVblockand negativeinotropy. 5

6. iOn Channel aSSayS heRg p2rx7 nav1.5 kv7.5 kv7.1/minK kv7.5/ Kv7.3 kv4.3 CiPA kv1.1 Cipa Kir2.1 kv1.2 Cav1.2 kv1.4 nav1.4 kv1.6 nav1.6 kv2.1 nav1.7 naChRa7 kv1.3 Gaba alpha 1, beta 3, gamma 2 kv1.5 Gaba alpha 5, beta 3, gamma 2 heag1 Gaba alpha 3, beta 3, gamma 2 tRaak Glu1 flop stim/orai Glur1flop /Aequorin Gaba alpha 2, beta 3, gamma 2 Glur1 / Glur2 Flip / Aequorin tRpv1 Glur5 / Aequorin aSiCt Glur6 hl-1 cells Glur6 / Aequorin Neuro2A cells (piezo) Glur6 / Aequorin PC12 cells (piezo) Glur5 / Glur6 / Aequorin Ins1 cells (glucose sensitive potassium channels) Glur5 / KA-2 / Aequorin Glur6 / KA-2 / Aequorin te671 (endogenous nAch-r with alpha1, beta1, gamma, delta) Glur5 / KA-1 / Aequorin Glur6 / KA-1/Aequorin Providing ion channel data for the FDA and the CiPA initiative J Pharmacol Toxicol Methods. 2016 Sep-Oct;81:251-62. An evaluation of 30 clinical drugs against the comprehensive in vitro proarrhythmia assay (CiPA) proposed ion channel panel. Crumb WJ Jr, Vicente J, Johannesen L, Strauss DG. 6

7. native CaRdiaC iOn Channel aSSayS ina role: Cell type: temperature: 23-25oC (INa cannot be reliably recorded at physiologic temperatures) Positive control: TTX ina (sodium current) isresponsibleforconductionintheheart. human atrial or ventricular myocytes ht: Manual: GlP: forscreeningpurposes IC50: 3-4 concentrations (n=2) foradetailedanalysis IC50: 6 concentrations (n=5) forregulatorysubmission IC50: 6 concentrations (n=5) iCa role: Cell type: temperature: 35-37oC Positive control:Nisoldipine iCa (calcium current) playsanimportantroleinthecontractionoftheheart human atrial or ventricular myocytes ht: Manual: GlP: forscreeningpurposes IC50: 3-4 concentrations (n=2) foradetailedanalysis IC50: 6 concentrations (n=5) forregulatorysubmission IC50: 6 concentrations (n=5) ik1 role: Cell type: temperature: 35-37oC Positive control:Ba++ ik1 (inwardly rectifying potassium current) helpssettherestingpotentialoftheheart. human atrial or ventricular myocytes ht: Manual: GlP: forscreeningpurposes IC50: 3-4 concentrations (n=2) foradetailedanalysis IC50: 6 concentrations (n=5) forregulatorysubmission IC50: 6 concentrations (n=5) 7

8. native CaRdiaC iOn Channel aSSayS Irole: Cell type: temperature: 35-37oC Positive control:4-AP ito (transient outward current) isarepolarizingcurrent human atrial or ventricular myocytes ito ht: Manual: GlP: forscreeningpurposes IC50: 3-4 concentrations (n=2) foradetailedanalysis IC50: 6 concentrations (n=5) forregulatorysubmission IC50: 6 concentrations (n=5) Irole: Cell type: temperature: 35-37oC Positive control:4-AP isus (sustained potassium current) isarepolarizingcurrent human atrial or ventricular myocytes isus ht: Manual: GlP: forscreeningpurposes IC50: 3-4 concentrations (n=2) foradetailedanalysis IC50: 6 concentrations (n=5) forregulatorysubmission IC50: 6 concentrations (n=5) First to provide mechanism for Qt prolonging effects of terfenadine in adult human cardiac myocytes Mol Pharmacol. 1995 Jan;47(1):181-90. Blockade of multiple human cardiac potassium currents by the antihistamine terfenadine: possible mechanism for terfenadine-associ- ated cardiotoxicity. Crumb WJ Jr, Wible B, Arnold DJ, Payne JP, Brown AM. 8

9. Why We uSe native human CaRdiaC myOCyteS the importance on proper ion channel physiology and pharmacology of accessory subunits, and a native intracellular milieu with all of the appropriate intracellular messengers has been well described. that is why at CytoBioscience, ion currents can be recorded from acutely isolated human atrial or ventricular myocytes. Isolated human cardiac myocyte assays •Mostrelevantspecies •Avoidsphenotypeissues ofiPScells • Noanimalsusedfortissue harvesting • Provenexperiencesince1996 there can be dramatic species differences in cardiac ion channel pharmacology. This figure illustrates the effect of a compound on INa recorded fromahumancardiacmyocyte,a guineapigmyocyte,andacanine myocyte. No species accurately duplicatedtheeffectseeninthe humancardiacmyocyte. see also: European Heart Journal Supplements (2001) 3 (Supplement K), K53–K63 Native and cloned ion channels from human heart: laboratory models for evaluating the cardiac safety of new drugs I. Cavero1 and W. Crumb2 9

10. iOn CuRRentS Can Be ReCORded fROm aCutely iSOlated human atRial OR ventRiCulaR myOCyteS. CaRdiaC aCtiOn pOtential Thecardiacactionpotential(AP)isadeflectionofvol- tage,lastingseveralhundredmilliseconds.TheAPis classicallydividedintophases(0-4).Phase0orthe upstrokeoftheactionpotentialisduetotherapidin- fluxofsodiumthroughthevoltagegated sodiumchannel(INa).Thisisfollowedbyphase1,or therapidphaseofrepolarization,whichistheresultof potassiumionsleavingthecellthroughthetransient outwardpotassiumcurrent(Ito).Nextistheplateauof theAP,orphase2,whichisinpartduetoaninfluxof calcium(ICa)followedbyphase3,orthemainphaseof repolarization,whichistheresultof aneffluxofpotassiumthroughavarietyofpotassium channels.Finallytherestingphase(phase4)whichis controlledinlargepartbythepotassiumchannelIK1. SpeCieS Cell type human adult atrial, ventricular, purkinje fibe human ipS ventricular or atrial myocytes human guinea pig atrial, ventricular, purkinje fiber canine atrial, ventricular, purkinje fiber rabbit atrial, ventricular, purkinje fiber 10

11. OuR team Of SCientiStS OffeRS extenSive expeRienCe … langendORff iSOlated heaRt aSSay Our team of scientists offers extensive experience leveraging the Langendorff Isolated Perfused Heart preparation to provide a complementary and comprehensive evaluation of possible cardiac responses and/or liabilities. Theisolated-heartpreparationisacost-effectiveandGLP-validatedrapidassessmentas- saywhichcansimultaneouslyevaluateelectrophysiological,mechanical,andmetabolic cardiacend-pointswithrepeatabilityandaccuracy.Notably,thispreparationisextremely effectiveinprovidingmillisecondaccuracyforassessingrepolarizationliabilitiesandpro- arrhythmicrisks(torsadogenicand/orre-entrant.) our experts have access to a large comparative database of positive and negative controls and can characterize the following: • Assessmentofelectrophysiologicalresponsesandliabilities-particularlychronotropy, dromotropy,andchangesintheduration,temporalstabilityandspatialheterogeneity ofventricularrepolarization(viatrans-mural/ventricularbipolarelectrogramsand monophasicactionpotentials) • Characterizationofdirect(load-independent)cardiacfunctional/mechanicaleffects (viaisovolumetricleftventricularpressures) • Quantificationofmyocardialmetabolicimbalances/loadingandcoronaryflow/ vascularresistance • Evaluationofdose-responseand/oruse-dependency • EstablishingofNOELandNOAELleveragingourion-channelexperiencetoprovide mechanisticunderstanding 11

12. in vivo aSSayS Glp compliant studies for ind submission non-Glp multiple Species: small(e.g., mice, rat, GP)to large(e.g., dog, sheep, pig , non human primate) COnSCiOuS pRepaRatiOnS • Telemetry(InvasiveandNon-Invasive;ECGandsystemic/ventricularpressures) • CardiacOutputandChronicLoad-IndependentCardiacFunction • Imaging(Echocardiography,CT,MRI,x-ray) • BloodSampling(PK) anaeSthetized pRepaRatiOnS SuRgiCal mOdelS and deviCe aSSeSSmentS COmpRehenSive methOdOlOgieS • CompletePhysiologicalEvaluation • ECG(rhythm,intervals,repolarization,beat-to-beatanalyses) • SystemicandCardiacHemodynamics • CardiacOutput,Fractionation/RegionalDistribution,andBloodFlowLoad- independentventricularfunctionandVentriculo-vascularcoupling • Myocardial/SystemicEnergeticsandOxygenation • Electrophysiology -His-bundle -programmed-electricalstimulation -“mapping” -conduction • Renal(GFR/RPF) • Baroreceptor/Autonomic • Respiratory(mechanics) • VascularFunctionHistology diSeaSe mOdelS • HeartFailure(chronicandacute) -Systolic(embolization,tachy-pacing,coronary-ligation,banding,drug-induced) -Diastolic(renoprivalhypertension,diabetes,genetic) • Arrhythmia(e.g.atrialfibrillation,sudden-death,torsadesdepointes,reperfusion) • Ischemiaand/orIschemia/Reperfusion • Pulmonaryhypertension(drug-induced,embolism,hypoxia) • Systemichypertension(renoprival,genetic,diet) • Diabetes • Embolism/Atherosclerosis(endothelialinjury,diet/drug-induced,Foltz) • Hemorrhagicshock • Pain(Cold-pressor) 12

13. With OuR in SiliCO SeRviCeS We Can help tO make SenSe Of yOuR data. in Silico SeRviCeS in-silico models of cardiac electrical activity have become very sophisticated in recent years, and allow a better understanding of the complex non-linear interactions between the different ionic currents and ion concentrations within the cell. OneoftheaimsoftheCiPAinitiativeistointegratedatafrompatchclampexperiments onarangeofimportantionchannelswithin-silicosimulationsofcardiacelectricalactivi- ty.CurrentlytheO’Hara&Rudymodel(seefigure1)isahotcandidatetobethemodelof choice,sinceitistheonlyavailablemodelofhumanventricularmyocytesbasedentirely onhumandata. Duetotheircomplexity,effectiveuseofthesemodelsandinterpretationofresultsrequires extensiveexperienceinthefield.HereatCytoBiosciencewearereadytosupportour customers both in in-vitro electrophysiology as well as in in-silico modeling. With our simulationswecanhelptomakesenseofyourdata. SeRviCeS OffeRed • Generaladviceonin-silicomodelingofcardiacelectricalactivity • Implementationofarangeofavailablecellularmodels • Diseasestatemodeling(e.g.hypertrophiccardiomyopathy,ischemia,long-QT syndromes).See figure 1 for an example. • Implementingionchannelblockbasedonelectrophysiologydata,plotting andinterpretationofresults Figure 1: Example simulation of the O’Hara & Rudy 20111 ventricular model in endocardial configuration in normal conditions and under hypertrophic cardiomyopathy conditions2. Application of 2nM Dofetilide was simulated by blocking 59% of IKr, and 17.4% of Ito. The physiological model action potentials were prolonged (black traces), but no arrhythmogenic activity was observed. In hypertrophic cardiomyopathy (red traces), the same percentage block of IKr and Ito causes alternans and early-after-depolarizations. 13

14. We develOp, qualify, and validate analytiCal methOdS fOR nOn -CliniCal dOSe fORmulatiOn analySeS. analytiCal SeRviCeS AdheringtoGlobalRegulatoryGuidelines,theCytoBioscienceAnalyticServicesDepart- mentofferscapabilitiestodevelop,qualify,andvalidateanalyticalmethodsinthesupport ofnon-clinicaldoseformulationanalysesforallin-vivo, ex-vivo,orin-vitroplatforms.Spe- cificexamplesincludediscovery,safetypharmacology,toxicology,genetictoxicology,and ionchannelstudies.Weofferpersonalizedattention,fastanalysis,andqualitydataand reportstoprovidetheresultsthatareneededtomeetprogramneedsanddeadlines. CytoBIosCIeNCe oFFers: Flexible scheduling Rapid study starts Personalized attention strict adherence to regulatory guidelines hPlC method development with: method validation/ qualification, assessing specificity, selectivity, accuracy, precision, and stability hPlC analysis for the determination of concentration and homogeneity in non-clinical dose formulations hPlC analysis for determination of purity 14

15. Contact us yOu’ve gOt queStiOnS? We’ve Got ANsWers. you’d like to learn more about CytoBioScience, our focus and our work? We have a dedicated team of well-trained and high-professionalized specialists located in our offices to meet our customers’ needs. We’re looking forward to talking to you. inteRnatiOnal headquaRteRS 3463MagicDrive,Suite120 SanAntonio,Texas78229.USA E-mail:info@CytoBioScience.com Tel:+1210-767–2727 OtheR lOCatiOnS CytoBioScience, inc. 1441Canalstreet NewOrleans,LA70112.USA Tel:+1210-767–2727 CytoBioScience, inc. 1500FirstAvenueNorth,SuiteL107 Birmingham,AL35203.USA Tel:+1210-767–2727 Cytocentrics BioScience Gmbh BuildingS20 Nattermannallee1 50829Cologne,Germany Tel:+4922116815273 15