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Chapter11TopoisomeraseItargeteddrugs-camptothecin200214ayLIB689171214HxCaDrugDiscLIB1113191215

DrugsAgainstCancer:StoriesofDiscoveryandtheQuestforaCureKurtW.Kohn,MD,PhDScientistEmeritusLaboratoryofMolecularPharmacologyDevelopmentalTherapeuticsBranchNationalCancerInstituteBethesda,Marylandkohnk@nih.govCHAPTER11Thecamptothecinstory:Froma“Happy”ChineseTree.In1960,theNationalCancerInstitute(NCI)beganasearchforanticancersubstancesinextractsfromplantsandanimals(socalled"naturalproducts").Thateffortwasaddedtotheongoingtestingoflargenumbersoforganicchemicalsforanticanceractivity.TheworkwasbeingcarriedoutundertheauspicesoftheNCI'sCancerChemotherapyNationalServiceCenter(CCNSC).EverysubstancetestedinthissystemreceivedanNSCnumbertocodeforitinthedatabase,whichhadinformationaboutchemicalstructure,origin,andtestresultsinanimalsandcancercelllines.Byfarthemostimportantdiscoveriesbythenaturalproductseffortwerecamptothecinandtaxol,bothofwhichwereisolatedfromplantmaterialbyMonroeWallandMansukhWaniattheResearchTriangleInstitute(RTI)inNorthCarolina(KohnandPommier,2000).Herewetellthestoryofcamptothecin;thestoryoftaxolistoldinChapter12.AccordingtoWani,whenhearrivedatRTI1962,therewasnothingthereexcept4walls,anditwasonlywhenthe5th'Wall'joinedhimatRTIthatthingsstartedtomove.WallandWaniworkedtogetherinalife-longcollaborationthatyieldedsomeofthemostimportantadvancesinthehistoryofcancerchemotherapy(Figure11.1).BeforecomingtoRTI,MonroeWallhaddirectedaprogramattheU.S.DepartmentofAgriculture(USDA)inasearchforplantmaterialsthatcouldbeusedasastartingpointforthesynthesisofcortisone,whichwasatthetimeinshortsupply.TheplantextractswerealsosenttoNCIfortestingagainstcancerinmice,andanextractfromtheChinesetreeCamptothecaaccuminata(Figure11.2)wasfoundtohaveanti-canceractivity.ThetreewasknownasXiShu,“Happytree.”

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AnticancersearchhoweverdidnotfitinUSDA'smandate,andWall'sdesiretofindtheanticancersubstanceinthoseextractshadtowaitafewyearsuntilhemovedtoRTI.In1963,WallandManistartedwith20kgofbarkandwoodfromCamptothecaaccuminata.Theymadeextractsfromthematerialandtestedthemforanticanceractivityinmice.Theytestedthemostactivesamplesateachpurificationstep.Itwasslowandpainstaking.Butby1966,theyhadpurecamptothecinandhaddetermineditsstructurebyx-raydiffraction(Figure11.5)(Wall,1966).

Figure11.1.MonroeE.Wall(right)andMansukhC.Wani(left),discoverersofcamptothecinandtaxol.

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Figure11.2.Camptothecaaccuminata(XiShu,“Happytree”)intheChengduBotanicalGarden-Chengdu,China.ItisnativenearthewarmhumidstreambanksinSouthernChinaandTibet.(Publicdomain,Wikipediacommons.)CamptothecinspecificallyinhibitstopoisomeraseI.Thefirstcluethatcamptothecintargetsatopoisomerase-likeenzymewasunknowinglyobtainedbySusanB.Horwitzin1973inanearlyobservationatatimewhentopoisomeraseshadnotyetbeendiscovered(HorwitzandHorwitz,1973)(Figure11.3).Thatwasyearsbeforethename"topoisomerase"wasinvented.Shehadexposedhumancancercellstocamptothecin,anovelanticancerdrug,andobservedthatthecell'sDNAstrandswerebrokenbythedrug.Whenthedrugwasremoved,theDNAstrandbreaksquicklyreversed.ItseemedthatthedrugcausedrepairableorreversibleDNAstrandbreaks.However,therewasanadditionalobservationthatwassobizarrethatitwasnotmentionedinherpaper,perhapsbecausethepapermightthennothavebeenacceptedforpublication.

About the same time, a similar finding in cultured cancer cells was independently reported by Ann Spataro and David Kessel (Spataro and Kessel, 1972). Also about the same time, Rajalakshmi and Sarma (Rajalakshmi and Sarma, 1973) reported that camptothecin broke DNA strands in the liver of treated rats and that the DNA was repaired surprisingly quickly. According to Dr. Silvio Parodi, who worked with D.S. Sarma under the supervision of Emmanuel Farber at Fels Research Institute in Philadelphia, they were looking at anti-neoplastic agents (especially of natural origin) for their potential carcinogenicity, testing for induction of chromosomal aberrations and

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sister chromatid exchanges when they observed the unusual DNA breakage and repair by camptothecin.

AfewmonthsbeforepublicationofSusan'spaper,Ivisitedherlaboratory,whichwasthenledbyArthurGrollmanatAlbertEinsteinMedicalCenterinTheBronx.IhadbeenstudyingDNAstrandbreakageandrepairbyvariousanticancerdrugs,andshethereforetoldmeaboutherfindingswithcamptothecin.Ithenaskedhowlongittookforthestrandstoberepaired.Aftersomehesitation,ArthurGrollmansaidthattherepairwasveryfast,sofast,eveninthecold,thattheycouldnotmeasureit.Iaskedhowthatcouldpossiblybe.Afterfurtherhesitation,Arthursaidhedidn'tknow,butthatmaybetherewasanenzymerighttherebythebreaksthatresealedthemimmediatelywhenthedrugwasremoved.ThatspeculationseemedsobizarrethatIcouldnotacceptit.However,itturnedoutthatGrollman'sspeculationwasrightonthemark,andtheresponsibleenzymewaslateridentifiedasthethenunknowntopoisomeraseI.SusanHorwitzhadobservedanewanticancerdrug-inducedmechanismofDNAbreakageandrepairthatwastohavemajorimpactoncancerchemotherapy.

Figure11.3.SusanBandHorwitz,workingatAlbertEinsteinMedicalCenter,discoveredthatcamptothecinproducedrapidlyreversibleDNAbreaks.ShealsodiscoveredthatanticancerdrugTaxolblockedmicrotubules(Chapter10).InviewoftheearlyevidencethatcamptothecincausedDNAbreaksandthatinhibitorsoftopoisomeraseIIcausedprotein-linkedDNAstandbreaks(seeChapter8),LeroyLiuandhiscolleaguestestedcamptothecinagainsttopoisomeraseII.TheyweresurprisedtofindthattherewasnoeffectontopoisomeraseII,butfoundthat

SusanBandHorwitz,

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camptothecininducedtopoisomeraseItoproducebothDNAstrandbreaksandDNA-proteincrosslinks(Hsiangetal.,1985;HsiangandLiu,1988).Indeed,JoeCoveyandChristineJaxelinmylaboratoryconfirmedthatcamptothecinproducestypicalprotein-linkedDNAstrandbreaks(Coveyetal.,1989).AsSusanHorwitzandArthurGrollmanhadsurmised,aDNA-associatedenzyme(lateridentifiedastopoisomeraseI)rapidlyreversedthestrandbreaks;theywouldhavebeenamazedtoknowatthetimethattheirpostulatedreversalenzymealsoproducedthebreaksinthefirstplace.TopoisomeraseIresolvestheover-andunder-twistedDNAduringtranscriptionandreplication.Figure10.1illustratesthefirstofthecell’stopologicalproblems.AsthepairedDNAstrandsseparateduringtranscriptionorreplication,theDNAtwistsarepushedaheadandwouldbecomebuncheduptoanextentthatstrand-separationcouldnotcontinue.Inthecaseoftranscription,thereisanadditionalproblembehindthebubbleofseparatedstrands.WhenthetranscribedRNAemerges(diagramBinFigure10.1),thecomplementaryDNAstrandsre-associate,buttherearenotenoughtwisttomakethestableonetwistper10base-pairs(Pommier,2013).TheproblemissolvedbytypeItopoisomerasesthattransientlycleaveoneDNAstrandandallowthestrandstoswivelandremovetheexcessiveordeficienttwistsastheDNAorRNAsynthesismachinerymarcheson.AfterswivelinghasremovedthestressontheDNAhelix,thetopoisomeraserapidlyresealsthebreak(Figure2).

Figure10.1.TheDNAtwistingprobleminreplication(DNAsynthesis,A)andtranscription(RNAsynthesis,B).Asthestrandsseparate,thetwistsarepushedaheadandwouldimpedefurtherstrandseparation.Theexcessiveordeficient

A.#DNA#replica-on#

Increasing*twist*

B.#Transcrip-on#

Increasing*twist*

decreasing*twist*

RNA*

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twistsareresolvedbytopoisomeraseI,whichisboundtoandmovesalongwiththereplicationandtranscriptionmachineries.Parallellinepairsrepresentdouble-strandedhelix.InA,theredlinesrepresentnewlysynthesizedDNA.InB,theredlinerepresentsnewlysynthesizedRNA.Thestrand-separationforksaremovingfromrighttoleft(fatbluearrows).

Figure10.2.Thetopologicalproblemresolvedbyatype1topoisomerases.TheenzymeresolvestheproblembybreakingoneDNAstrand,allowingtheotherstrandtopassthroughthebreak,andfinallyresealingthebreak.Theredstrandisbrokenandthebluestrandpassesthrough.Astheenzymebreaksthestrand,itgrabsholdofoneendviatheenzyme’styrosine(Y)attheactivesite((Pommier,2013);ACSChemicalBiology,permissionneeded).HowcamptothecincausesDNAdamagethatkillscancercells.AnearlycluetohowcamptothecinkillscellswasthatitblockedthecellcycleinSphase,andthedrugselectivelykilledcellswhentheywerereplicatingtheirDNA(Goldwasseretal.,1996).WesurmisedthatcellswereselectivelyblockedinSphasebecauseofcollisionsbetweenmovingreplicationforksandsitesontheDNAwheretopoisomerase1wastrappedbycamptothecin(Figure11.4).OurviewwasbasedonhowtopoisomeraseIoperates:itbindsDNAinfrontofmovingreplicationforksandcyclesthroughopeningandclosingofaDNAstrandbreak,soastoallowthestrandstoswivelandrelievetheaccumulatingsupercoilingoftheDNAhelix(Chapter8,Figure8.1).CamptothecinbindstopoisomeraseIwhilethelatterisboundtoDNAandhascuttheDNAstrand.Whenamovingreplicationforkcollideswithsuchastabilizedcomplex,itresultsinafreedouble-strandendthatlookstothecelllikeaDNAdouble-strandbreak(Figure11.4)(KohnandPommier,2000).WesuspectedthatcamptothecinstackedagainstDNAbasepairs(asshowninFigure11.4),basedonexperimentalfindingsthatwereanalogoustothoseweobservedfortopoisomeraseIIinhibitors;moreover,camptothecinpreferentiallytrappedtopoisomeraseatsiteswheretheenzymelinkedtoDNAataGnucleotide(Chapter8,Figure8.8)(Jaxeletal.,1991).AlthoughcellsactivelyreplicatingtheirDNAweremostsensitivetocamptothecin,absenceofDNAreplicationdidnotfullyprecludetoxicity,probablybecausetheRNAsynthesisprocess(‘transcription’)couldalsocollidewithtrappedcamptothecin-topoisomerase-DNAcomplexes.CollisionsduetoprogressofRNAsynthesis,

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however,producedalesserdegreeoftoxicity,perhapsbecauseenzymesassociatedwiththetranscriptionmachineryareableusuallytoclearthetracksforcopyingtheDNAtemplatestrand..MoredetailedinvestigationoftheTop1-camptothecininteractionidentifiedamutantTop1enzymesthathadachangeintheaminoacidatacriticalsiteintheprotein.ThemutantenzymefunctionedasitshouldinrelievingstressfulDNAtwistsbutdidnotproduceDNA-Top1trappedcomplexeswithcamptothecin.ThepointhereisthatcellshavingonlythismutantformofTop1werenotkilledbycamptothecin.ThatfindingwasevidencethatthetrappedTop1-DNAcomplexeswereinfactwhatwaskillingthecells,Itwasindependentoftheinhibitionofstranduntwisting,andthereforewasattributabletothecollisioneventperse(Andohetal.,1987)(Guptaetal.,1988).Thus,theverytransientcamptothecin-inducedDNAbreakage,originallyobservedbySusanHorwitzandArthurGrollman,wasfoundtobeduetoaneffectontopoisomeraseI(Top1)(Hsiangetal.,1985)(Coveyetal.,1989).AswiththetopoisomeraseIItargeteddrugs,DNAstrandbreaksandDNA-proteincrosslinkswereproducedinequalnumbers,consistentwithoneproteinboundconsistentlytooneendofeachDNAstrandbreak(Matternetal.,1987).ThecovalentassociationofTop1ateachcamptothecin-inducedDNAbreakwasthenconfirmedbyHsiangandLiu(HsiangandLiu,1988).PorterandChampouxthenobtainedevidencethatthetrappingoftheTop1-DNAbreakswasduetoreductionbycamptothecinoftherateatwhichthebreaksreseal(PorterandChampoux,1989).ThesestudiesestablishedtheessentialfeaturesofhowcamptothecintrapsDNA-Top1.Laterstudies,however,disclosedthattheformationofthedisastrousDNAdouble-strandendshowninFigure11.4incellstreatedwithatopoisomeraseinhibitorcanbeavoidedifthedrugconcentrationisnottoohigh.WhenthegrowingendofareplicatingDNAencountersadrug-inducedblock,thegrowingreplicationfork,insteadofproceedingintotheblockedregion,cantemporarilyreverse,asshowninFigure11.5(RayChaudhurietal.,2012).Figure11.6showsanelectronmicroscopeimageofareversedreplicationfork.Thenewunderstandinginthe1980’sofhowcamptothecinworksgreatlyrevivedinterestintestingthedrugoncancerpatients;camptothecinandrelatedtopoisomeraseIinhibitorshavesinceassumedanimportantroleincancerchemotherapy.Thereversalofthereplicationforkismediatedinpartbypoly(ADPR)polymerase(PARP)(RayChaudhurietal.,2012)(seeChapter19).(Miaoetal.,2006)–tdp1-scan1

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Figure11.4.Howanencounterbetweenamovingreplicationprocessandacamptothecin-blockedtopoisomeraseIcomplexgeneratesapotentiallylethalDNAdouble-strandend,asweenvisioneditin1993(Pommieretal.,1994).(CellsthatarenotintheprocessofreplicatingtheirDNA,however,arestillsomewhatsensitivetocamptothecin,becauseofanalogousencountersoftrappedtopoisomeraseIbyatranscriptionprocess(Bendixenetal.,1990).)

Figure11.5.ReversalofreplicationforkwhenreplicatingDNA(brownlines)encountersablock,suchasproducedbyatopoisomeraseinhibitor.Ifthedrugconcentrationisnottoohigh,thereplicatingstrandscanreversetemporarilyuntiltheblockisremoved.

Replication complex

Block

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Figure11.6.Electronmicroscopeimageofreversedreplicationfork(RayChaudhurietal.,2012).Notethe4DNAdouble-helicesemergingfromthereversalpoint(arrow).(Permissionneeded.)Earlyclinicaltrialsofcamptothecin.Aspreludetotrialsofcamptothecinincancerpatients,testinginanimalsshowedthatadose-limitingtoxicitywasdamagetothelowerintestinaltract.Thecellsoftheinnerliningoftheintestinesmultiplyrapidlyinordertorenewcellsthatnormallyarecontinuallysluffedoff.Incancerpatients,however,thedose-limitingtoxicitywassuppressionofbloodcellproductioninthebonemarrow(Gottliebetal.,1970).Nevertheless,therapidlydividingcells,bothintheintestinesandinthebonemarrow,areparticularlysensitivetocamptothecin.Oneoftheproblemswiththeearlyclinicaltrialsofcamptothecinwasthattheyusedthesodiumsaltform(Figure11.7,right),whichisinactiveanditsconversiontotheactivelactoneform(left)inpatientsiserratic.Thecamptothecinlactoneistheactiveform,butitisnearlyinsolubleandthereforedifficulttoprepareforclinicaluse.(Thesolubilityproblemwaslatersolvedbyencapsulatingtheinsolublecamptothecinlactoneingelatincapsulesfororaladministration).However,thesodiumsaltissolubleandreadilyadministered.Itwasthereforeusedintheearlystudieswhenitslackofactivitywasyetunknown(Muggiaetal.,1996).Theearlyclinicalexperiencewithcamptothecinwasdiscouraging,andthereforetheclinicaltrialswerestopped.Camptothecinstudieswereresumed15yearslaterwhenitsactionontopoisomeraseIwasdiscovered(Chapter8).Developmentofcamptothecinasananticancerdrugthenresumedwithrenewedintensity,althoughthelapsof15yearswasunfortunateforadrugthatwastobecomeveryusefulforanticancertherapy.

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Figure11.7.Chemicalstructureofcamptothecin.Theactiveformofcamptothecinhasa“lactone”structureintheEring(left).Underalkalineconditions,thelactoneringopenstoformthesodiumsalt(right),whichisinactive.Undermildacidicconditions,thesodiumsaltslowlyconvertstotheactivelactoneform.NotablealsoisthatthenaturalactiveformhasitsOHgroupatposition20pointingup,whereastheisomerwhoseOHpointsdownisinactive.Thusthe3-dimensionalstructurearoundposition20hastobejustrightforcamptothecintobindtotheTop1protein(seechapter8).Newdrugsbymodifyingthestructureofcamptothecin.In1989,wecollaboratedwithMonroeWallandMansukhWaniintestingalargenumberofmodifiedcamptothecinsfortheiractivityagainsttopoisomeraseI(Jaxeletal.,1989)(KohnandPommier,2000).Theresultsshowedwherethecamptothecinmoleculecouldbemodifiedtoincreaseitspotencyandindicatedwheremodificationsabolishedactivity.WefoundoutwherethecamptothecinmoleculemustremainunobstructedinordertofitintoitsbindingsiteonthetopoisomeraseIprotein,andwhereatomscouldbeaddedwithoutlossofactivity.Forexample,addinganNH2groupatposition9ontheAringincreasedactivity,whereasaddinganNH2groupatposition12destroyedactivity.Thus,position12hadtoremainunobstructedtoallowcamptothecintofitwellintoitsbindingsiteontopoisomeraseI.Positions10and11werefreeformakingsmalladditions.Infact,addinganOHgroup,especiallyatposition10,substantiallyincreasedcamptothecinpotency(Jaxeletal.,1989).Amongthemodifiedcamptothecinsweexamined,oneofthemostpotenthadamethylenedioxy(-O-CH2-O-)groupaddedtoforma5-memberedringnexttotheAring(Figure11.8)(O'Connoretal.,1990;O'Connoretal.,1991).Althoughthiscompoundwasnotpursuedfordevelopmentatthattime,itwaslaterrediscoveredandcalled"FL118"(Lingetal.,2012;Lingetal.,2015).

CAMPTOTHECIN+(LACTONE)+ CAMPTOTHECIN+(SODIUM+SALT)+

Na++

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Figure11.8.10,11-methylenedioxycamptothecin,amodifiedcamptothecinhavingincreasedpotencyforinhibitionoftopoisomerase1(Jaxeletal.,1989).Theadditiontothecamptothecinmoleculeiscircledred.Topotecanbecamethemostfrequentlyusedcamptothecinincancertherapy.Ourstructure-activityfindings(Jaxeletal.,1989)helpedtodesignthemodifiedcamptothecin,"topotecan",whichbecamecommonlyusedincancertreatment.Topotecanhasapositivelychargedmethylaminogroupaddedatposition9andanOHgroupaddedatposition10(Figure11.9).Thepositivelychargedgroupsolvedthesolubilityproblem;itsplacementatposition9wasinaccordwithourfindingthatadditionscouldbemadeatthispositionwithoutinterferingwiththeabilityofthedrugtoblocktopoisomeraseI.WehadalsofoundthataddinganOHgroupatposition10,whichisthecasefortopotecan,wouldincreasethepotencyofthedrug.Topotecanwasrelativelyeasytomakebychemicalmodificationofcamptothecin,anditwashighlypotentagainstexperimentaltumorsinanimals,aswellaseffectiveagainsttopoisomeraseIincancercells(Kingsburyetal.,1991).

Figure11.11.Topotecan,amodifiedcamptothecinbecameusedincancertherapy.TheN-containinggroupaddedatposition9becomespositivelycharged,andthereforeimprovesthesolubilityofthedrug,sothatitcanreadilybeadministeredtopatients.TheOHgroupaddedatposition10increasedthepotencyofthedrug.

10#

11#

9"

10"

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IrinotecanAnothermodifiedcamptothecin,irinotecan,alsobecamecommonlyusedintherapy.ItwasapprovedbytheU.S.FoodandDrugAdministrationin1996forthetreatmentofcoloncancer;itwasalsoactiveagainstseveralothertypesofcancer.Irinotecanisa"pro-drug":itisnearlyinactiveuntilacarboxyesteraseenzyme,presentinliverandothertissues,cutsoffaninactivatingside-chainfromthemolecule(Figure11.10A)(Rameshetal.,2010).Whencombinedwithotherdrugs,suchas5-fluorouracilandoxaliplatin,itbecameakeydrugforthetreatmentofmetastaticcolorectalcancer,anditwasalsousefulagainstseveralothertypesofcancer(Fujitaetal.,2015).

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Figure11.10.A.Chemicalstructureofirinotecan.Thesidechainontheoxygenatposition10conferswater-solubility,butinactivatesthedrug.Thedrugisactivatedbyandenzymepresentintissuesthatcleavesofthebondindicatedbytheredarrow(Rameshetal.,2010).ThatleavesanOHgrouponposition10,whichincreasesthepotencyofthedrug(Jaxeletal.,1989). B.Chemicalstructureafteradditionofaglucose-like(glucuronide)unitbytheUGTenzyme,whichinactivatesthedrug(Rameshetal.,2010).AbsenceofthisenzymeallowstheamountofavailableactivedrugtoincreasetohigherlevelsandtherebymakespatientswholackactiveUGTunusuallysensitivetothedrug.Irinotecanproducesunusuallyseveretoxicityinsomepatients.Extensivestudieswerecarriedouttofindoutwhythatisthecase.Iftheunusuallysensitivepatientscouldbeidentified,theirdrugdosecouldbereducedtoasafelevel.ThestudiesrevealedthatafrequentcauseoftheunusualsensitivitywasaparticularisoformofagenecalledUGT1A1thatsensitivepatientshadintheirgenome.ThisgenewasfoundtocodeforanenzymecalledUDP-glucuronosyl-transferase(UGT),whosefunctionwillbeexplainedshortly.AmongtheseveralgenesthatcodeforUGTenzymes,themosttroublesomeformwasUGT1A1*28.PeoplewhohadonlythatisoformoftheUGT1A1genewerehighlysensitivetoirinotecan.Thereasonforthat

10#

A"

B"

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wasthattheUGTenzymemadebythatisoformwasnearlyinactive(Schulzetal.,2009)(FujiwaraandMinami,2010).AccordingtoDr.SilvioParodi,UGT(UDP-glucuronosyltransferase)isacytosolicglycosyltransferasethatcatalyzesthetransferoftheglucuronicacidcomponentofUDP-glucuronicacidtoasmallhydrophobicmolecule.Thisisaglucuronidationreaction.ThereactioncatalyzedbytheUGTenzymeinvolvestheadditionofaglucuronicacidmoietytoavarietyofbiologicallyactivecompoundsfoundinnature.Tounderstandallthat,wehavetoknowwhattheactiveUGTenzymedoes.Afteririnotecanhasbeenactivatedbycuttingoffthesidechainfromposition10(Figure11.10A),UGTinactivatesitagainbyaddingaglucuronideunit(Figure11.10B).WithoutactiveUGT,therefore,thelevelofactiveirinotecanwaselevatedtounusuallyhighlevelsafterthecustomarydoseofthedrug(Schulzetal.,2009)(FujiwaraandMinami,2010).ThesolutiontotheirinotecandosageproblemthereforewastodeterminetheUGTstatusofthepatientandadjustthedrugdosageaccordingly.Aremarkablemodificationofirinotecan,calledetirinotecanpegol,wasdesignedthatreducedtoxicityandincreasedanti-tumorpotencyinmicebyslowlyreleasingtheactivetopoisomeraseIinhibitoroverlongperiodsoftime(Figure11.11).Thestructurewasdesignedtolinkirinotecantolongpoly(ethyleneglycol)chainsinamannerthatkeptthedruginactiveandtoslowlyandspontaneouslyreleaseitinitsactiveform(Hochetal.,2014).Etirinotecanpegolwasmoreeffectivethanthebareirinotecaninsuppressingthegrowthoftumorsinmice(Figure11.12),andclinicaltrialsofthispromisingdesignerdrugwerebegun(Alemany,2014)(Jamesonetal.,2013;Lopez-MirandaandCortes,2016).

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Figure11.11.Molecularstructureofetirinotecanpegol,inwhichirinotecanmoleculesweretetheredtotheendsofpoly(ethyleneglycol)chains.Thelinkerslowlyhydrolysestoreleaseactiveirinotecan(Hochetal.,2014)(permissionneeded).

Figure11.12.Increasedeffectivenessofetirinotecanpegol(EP)relativetobareirinotecan(IRN)againsthumanlungcancercellsgrowingastumorsinimmune-deficientmice(Hochetal.,2014).Verticalaxis:tumorvolume;horizontalaxis:timeaftertreatment(arrowsshowtimesofEPinjection).EPinhibitedtumorgrowthfor

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amuchlongertimethandidIRN.Similarresultswerereportedwithseveralcelllinesfromothertypesofhumancancer.(Permissionneeded.)Anotherwaytomakeirinotecanmoreeffectivewastoincorporatethedrugintinymicroscopic-sizedlipidglobulescallednanoliposomes,fromwhichthedrugwasslowlyreleased.Inaddition,theideawasthatthenanoliposomeswouldbesmallenoughtoexitfromthetumor'sabnormalbloodvessels,whilebeinglargeenoughtoberetainedinnormalbloodvessels.Thatwouldselectivelydeliverthedrugtothetumorandreducetoxiceffectstonormaltissues.Anotherfactorwouldbethatdrugwithinthetumortissuewouldonlyslowlybeflushedout,becauseofthepoorlymphaticdrainagethatiscommonintumors.Nanoliposomalirinotecan("nal-irinotecan")hasalreadybeenapprovedforclinicaluse(Ko,2016).ReferencesAlemany,C.(2014).Etirinotecanpegol:developmentofanovelconjugated

topoisomeraseIinhibitor.Currentoncologyreports16,367.Andoh,T.,Ishii,K.,Suzuki,Y.,Ikegami,Y.,Kusunoki,Y.,Takemoto,Y.,andOkada,K.

(1987).Characterizationofamammalianmutantwithacamptothecin-resistantDNAtopoisomeraseI.ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica84,5565-55611.

Bendixen,C.,Thomsen,B.,Alsner,J.,andWestergaard,O.(1990).Camptothecin-stabilizedtopoisomeraseI-DNAadductscauseprematureterminationoftranscription.Biochemistry29,5613-56111.

Covey,J.M.,Jaxel,C.,Kohn,K.W.,andPommier,Y.(1989).Protein-linkedDNAstrandbreaksinducedinmammaliancellsbycamptothecin,aninhibitoroftopoisomeraseI.Cancerresearch49,5016-5022.

Fujita,K.,Kubota,Y.,Ishida,H.,andSasaki,Y.(2015).Irinotecan,akeychemotherapeuticdrugformetastaticcolorectalcancer.Worldjournalofgastroenterology21,12234-12248.

Fujiwara,Y.,andMinami,H.(2010).Anoverviewoftherecentprogressinirinotecanpharmacogenetics.Pharmacogenomics11,391-406.

Goldwasser,F.,Shimizu,T.,Jackman,J.,Hoki,Y.,O'Connor,P.M.,Kohn,K.W.,andPommier,Y.(1996).CorrelationsbetweenSandG2arrestandthecytotoxicityofcamptothecininhumancoloncarcinomacells.Cancerresearch56,4430-4437.

Gottlieb,J.A.,Guarino,A.M.,Call,J.B.,Oliverio,V.T.,andBlock,J.B.(1970).Preliminarypharmacologicandclinicalevaluationofcamptothecinsodium(NSC-100880).CancerchemotherapyreportsPart154,461-470.

Gupta,R.S.,Gupta,R.,Eng,B.,Lock,R.B.,Ross,W.E.,Hertzberg,R.P.,Caranfa,M.J.,andJohnson,R.K.(1988).Camptothecin-resistantmutantsofChinesehamsterovarycellscontainingaresistantformoftopoisomeraseI.Cancerresearch48,6404-6410.

Hoch,U.,Staschen,C.M.,Johnson,R.K.,andEldon,M.A.(2014).Nonclinicalpharmacokineticsandactivityofetirinotecanpegol(NKTR-102),along-acting

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topoisomerase1inhibitor,inmultiplecancermodels.Cancerchemotherapyandpharmacology74,1125-1137.

Horwitz,S.B.,andHorwitz,M.S.(1973).EffectsofcamptothecinonthebreakageandrepairofDNAduringthecellcycle.Cancerresearch33,2834-2836.

Hsiang,Y.H.,Hertzberg,R.,Hecht,S.,andLiu,L.F.(1985).Camptothecininducesprotein-linkedDNAbreaksviamammalianDNAtopoisomeraseI.TheJournalofbiologicalchemistry260,14873-14878.

Hsiang,Y.H.,andLiu,L.F.(1988).IdentificationofmammalianDNAtopoisomeraseIasanintracellulartargetoftheanticancerdrugcamptothecin.Cancerresearch48,1722-1726.

Jameson,G.S.,Hamm,J.T.,Weiss,G.J.,Alemany,C.,Anthony,S.,Basche,M.,Ramanathan,R.K.,Borad,M.J.,Tibes,R.,Cohn,A.,etal.(2013).Amulticenter,phaseI,dose-escalationstudytoassessthesafety,tolerability,andpharmacokineticsofetirinotecanpegolinpatientswithrefractorysolidtumors.Clinicalcancerresearch:anofficialjournaloftheAmericanAssociationforCancerResearch19,268-278.

Jaxel,C.,Capranico,G.,Kerrigan,D.,Kohn,K.W.,andPommier,Y.(1991).EffectoflocalDNAsequenceontopoisomeraseIcleavageinthepresenceorabsenceofcamptothecin.TheJournalofbiologicalchemistry266,20418-20423.

Jaxel,C.,Kohn,K.W.,Wani,M.C.,Wall,M.E.,andPommier,Y.(1989).Structure-activitystudyoftheactionsofcamptothecinderivativesonmammaliantopoisomeraseI:evidenceforaspecificreceptorsiteandarelationtoantitumoractivity.Cancerresearch49,1465-14611.

Kingsbury,W.D.,Boehm,J.C.,Jakas,D.R.,Holden,K.G.,Hecht,S.M.,Gallagher,G.,Caranfa,M.J.,McCabe,F.L.,Faucette,L.F.,Johnson,R.K.,etal.(1991).Synthesisofwater-soluble(aminoalkyl)camptothecinanalogues:inhibitionoftopoisomeraseIandantitumoractivity.Journalofmedicinalchemistry34,98-107.

Ko,A.H.(2016).Nanomedicinedevelopmentsinthetreatmentofmetastaticpancreaticcancer:focusonnanoliposomalirinotecan.Internationaljournalofnanomedicine11,1225-1235.

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