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Chapter5.Themethotrexatestory–version200218dgDrugsAgainstCancer:StoriesofDiscoveryandtheQuestforaCureKurtW.Kohn,MD,PhDScientistEmeritusLaboratoryofMolecularPharmacologyDevelopmentalTherapeuticsBranchNationalCancerInstituteBethesda,Marylandkohnk@nih.govCHAPTER5:Themethotrexatestory:folicacidanalogsDiscoveryofmethotrexateasananti-leukemiadrugAcuteleukemiawasrelentlessandinvariablyfatal,andtherewasnowayofevenslowingdownthedisease.Thatterribledisease,oftenofchildren,iscausedbyabnormalwhitebloodcellsgrowingunchecked:theyovergrowthebonemarrowandblocknormalbloodcellproductionthere.Theresultisdepletionofredbloodcellswithconsequentanemia,dearthofnormalwhitebloodcellsthatareneededtofightinfections,andreductioninplateletsneededtopreventbleeding.InJune1948,just2yearsafterGoodman,Gilmanandtheircoworkersreportedthelymphomatumor-meltingeffectofnitrogenmustard(Goodmanetal.,1946)(seeChapter1),SidneyFarberandhiscoworkersatHarvardMedicalSchoolandTheChildren'sHospitalinBostonreportedthataminopterin,ananalogandantagonistoffolicacid,wasabletoslowtheprogressofchildhoodleukemia(FarberandDiamond,1948)(Figures5.1).Thatwasthesecondbreakthrough,afternitrogenmustard,thathastenedtheeraofcancerchemotherapy.Althoughitwasnotacure,itdidsetthestageforacure.Aminopterinwasachemicallymodifiedfolicacidthatwasknowntoinhibittheactionsoffolicacid.ThisinhibitionimpairedtheproductionofbuildingblocksforthesynthesisofDNAandRNA.Consequently,thedrugimpairedtheabilityofcellswereunabletogrowanddivide.Farberhadfolloweduponareportin1947fromLederleLaboratoriesinPearlRiver,NewYorkthatfolicacidantagonistssuppressedwhitebloodcellcountsinrats.A

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modeststretchoftheimaginationsuggestedthatsuchanti-folatedrugsmightimpairthegrowthofleukemicbloodcells(Franklinetal.,1947).Farberbegancautiouslytreatingchildreninthelaststagesofthediseasewithmodifiedformsoffolicacid.Aftersomeencouragingresults,Farberselectedaminopterinforthefurtherstudies,becauseitwasthemostpotentfolicacidantagonistavailable.Totheinvestigators'surpriseanddelight,someofthechildrenhadaremarkableresponse:theirsymptomsimprovedandtheirleukemiacellsdisappeared(FarberandDiamond,1948).Forashorttime,itevenseemedasiftheymightbecured.Butwithinafewmonthsleukemiacellsbegantogrowagain,andthosenewlygrowingleukemiacellsdidnotrespondedtothedrug.Similartemporaryresponsesweresoonreportedalsoinadultpatientswithacuteleukemia(Dameshek,1949).Inadditiontoaminopterin,thelattertrialsusedanotherfolateantagonist,amethopterin,whichcametobecalledmethotrexate,andwhichbecameamainstayofcancertherapy.Damesheklikenedacuteleukemiatoawildfire,which,althoughdampenedbyaminopterin,continuestosmolderandmaysuddenlylightupagain(Dameshek,1949).Thetemporaryresponsesofchildhoodacuteleukemiatothe"antifols"wereimpressiveandbeyondpreviousexperience.However,aminopterinormethotrexate,usedbyitself,wasfarfromacure.Therootsoftheantifoldiscoveryhowevercanbetracedfurtherbacktothe1930'sandearly1940's,whenresearchersfoundthatafolicaciddeficiencyoftencausedanemia(HoffbrandandWeir,2001).Thebonemarrowofsomeoftheanemicpatientscontainedunusualenlargedcellsthattheythoughtresembledleukemiacells.Theresearchersthereforethoughtthatleukemiamightresultfromafolicaciddeficiency.Thiswasincorrect,however,becausethoseenlargedcellswereabnormalprecursorsofredbloodcells,notleukemicwhitebloodcells.Eventhoughtheconjecturewaswrong,itledtoamajorbreakthrough.Followinguponthaterroneousidea,HenleandWelchtreatedaleukemiapatientwithfolicacid,thinkingthatthattheleukemiawascausedbyafolicaciddeficiency.Insteadofslowingthedisease,however,folicacidcausedittoprogressfaster.Well,theythought,iffolicacidspeededupthedisease,maybefolicaciddeficiencywouldslowitdown.Indeed,whentheytreatedanotherleukemiapatientwithacrudefolicacidantagonist,therewasadramaticreductioninthenumberofleukemiacellsintheblood.HenleandWelchpublishedthisobservationinaverybriefreportin1948(HeinleandWelch,1948).Itwasthefirstcluethatfolicacidantagonistscouldsuppresstheprogressofleukemia.Thatbriefreport,spurredchemistsatLederleLaboratoriestosynthesizenewfolicacidantagonist.Themostpotentofthesewasaminopterin,whichwasthedrugSidneyFarberusedinhislandmarkfindingsinthetreatmentofchildhoodleukemia--whichwasalsopublishedin1948,showinghowquicklyapreliminaryobservationledtoasubstantialclinicalresult.

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Itwasnotyetacure,however,becausethepatientsinevitablyrelapsedandtheirleukemiathennolongerrespondedtothedrug.Thisexperiencehoweverprovidedafoundationfortheeventualcureofacuteleukemiainchildren.

Figure5.1.SidneyFarber(1903-1973),discovererofaminopterinandmethotrexateaseffectivedrugsforthetreatmentofacuteleukemiainchildren.Althoughtheydidnotcure,thedrugsdidtemporarilyshutdownthediseaseandprolonglife.Aminopterin'sactionagainstchildhoodleukemiawassoonconfirmedandextendedtoleukemiainadults,aswellassolidtumors,suchasbreastcancer(reviewedbyFarberandbyDameshekin1949(Farber,1949)(Dameshek,1949)).Thespeedofthisprogressindiscoveryandclinicalapplicationisnotable,especiallywhencomparedwiththedelaysanddifficultiesthatnewtherapiesnowoftenencounter(DeVitaJr.,2015).Still,temporaryremissionsinthoseearlystudieswereachievedinonlyafractionofpatients,andatthecostofsometimesseveretoxicity.Ataboutthesametime,ChesterStockandAbrahamGoldinandtheircolleaguesshowedthataminopterininhibitedthegrowthofmalignanttumorsinmice(Schoenbachetal.,1949;Sugiuraetal.,1949)(Figure5.2).Moreover,theeffectofthedrugwaspreventedbyfolicacid,whichsupportedtheideathataminopterindidinfactinhibitthetumorbycompetingwithfolicacid(Goldinetal.,1949).Aminopterindiffersfromfolicacidonlyinthatanoxygenatomisreplacedbyanaminogroup(Figure5.3).Itisnowknownthataminopterinormethotrexatecompetewithanactiveformoffolicacidforbindingtotwocriticalenzymes,aswillbeexplainedlaterinthischapter.

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Figure5.2.Effectofaminopterinonamousetumor(sarcoma180).Left,beforetreatment;right,aftertreatment.Aftertreatment,thetumorcellsweredyinganddisintegrating(240X)(Sugiuraetal.,1949).AbrahamGoldinandhiscoworkersatNCIfoundthatamethopterin(methotrexate)hadabettertherapy-versus-toxicityratiothanaminopterin.Therefore,in1956,methotrexatereplacedaminopterinintreatmentofpatients.Aminopterinandmethotrexatearechemicallyandpharmacologicallyverysimilar;howeveritseemsthatthetwodrugsmayneverhavebeencomparedhead-to-headinhumanpatients(Bertino,1993).Interestingly,methotrexatehadbetterantitumorproperties(inanimals),despitebeingmuchlesspotent(ahigherdoeswasneeded)thanaminopterin(Fergusonetal.,1950).Bothaminopterinandmethotrexatekilledmostoftheleukemiacells,butalsodepletedthebonemarrowofthenormalredbloodcells,whitebloodcells,andplateletsneededtopreventanemia,fightinfection,andpreventbleeding(Thiersch,1949).Therefore,thenormalbonemarrowwasgiventimetorecoverbetweentreatments.Amajorsteptowardtheeventualcureofchildhoodleukemiawasthedevelopmentofplatelettransfusion,whichpreventedbleedingduringthetimerequiredforthebonemarrowtorecoverThiscriticaldevelopmentwasspearheadedbyEmilJFreireichattheNationalCancerInstitute.Methotrexatebyitselfproducedremissionsthatonlylastedseveralmonthstoaboutayear.Lifewasprolonged,buttheleukemiainvariablyrecurredandnolongerrespondedtothedrug.Cancerresearchershoweverwererelentlessintheirquesttocurethedisease;itwasalongstruggle,butoverthenextthreedecadestheysucceededindoingso.Folicacidantagonistswereanessentialpartofthestory,buteventualsuccessrequiredthecarefuldesignoftherapyusingmulti-drug

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combinations,aswellasplatelettransfusionsandbonemarrowimplants(DeVitaJr.,2015).Theroadtothecureofchildhoodleukemiawasalonganddifficultstruggle.Somecliniciansinthe1940'sand1950'sfeltthatthechildrenshouldbeallowedtodieinpeace,ratherthanbeingsubjectedtotheadditionaldiscomfortsofdrugtoxicitiesandthepainofbonemarrowaspirationsthatwereneededtogagetheeffectsofthedrugs.Evenin1957,whenIarrivedatNCIandservedonthechildhoodleukemiaward,someofmyfellowClinicalAssociatesfeltthatwayandatleastoneofmyclosefriendsrefusedtoserveonthecancerwards,becausehefeltthatsomeoftheresearchwasunethical.However,ifleftuntreated,thesechildrenwereallfatedsoontodieoftheirdisease,andmanyparentsfeltthatanythingwasworthatry.Wedidsucceedintemporarilysuppressingthediseasewithmethotrexate,asFarberhaddescribed,aswellaswithotherdrugsthatwerebeingtried.MyclinicalassociatecolleaguesontheNCIcancerwardsinthelate1950'showeverwouldhavebeensurprised,asImyselfwas,thattheclinicalstudiesofthoseearlydayswerethebeginningofapaththatdidleadtoacure.

Figure5.3.Simplemodificationsoffolicacidyieldtheanticancerdrugsaminopterinandamethopterin(nowcalledmethotrexate).Placinganaminogroup(NH2)inplaceoftheoxygenonthepteridineringoffolicacidyieldedaminopterin;furtheradditionofthemethyl(CH3)group(encircledred)yieldedmethotrexate.ThereplacementofthepteridineoxygenbyanNH2group,causedthemoleculetobecomeanantagonistoffolicacid:itinhibitedtheactionsoffolicacidandputamonkeywrench(theEnglishmightsay“spanner”)intothemechanismswherefolicacidiscritical.

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MethotrexatecureschoriocarcinomaOnly2yearsafterAbrahamGoldin'sdiscoveryofthesuperioreffectivenessofmethotrexate,RoyHertzandhiscolleaguesatNCIreportedthatmethotrexatewasremarkablyeffectiveagainstchoriocarcinoma,arapidlyfatalcancerarisingfromembryonictissuesoftheplacentainpregnantwomen(Figure5.4)(Hertzetal.,1961;Hertzetal.,1956;Lietal.,1958).Methotrexate'sdramaticcureofmanycasesofchoriocarcinomawassoonconfirmedbyJamesHollandattheRoswellParkMemorialInstituteinBuffalo,NewYork(Holland,1958).Methotrexate,givenintheappropriatedoseschedule,curedmostofthepatients,evenifthetumorhadalreadymetastasized(Hertzetal.,1964).Thereportedcureofametastaticcancerastoundedmanycancerresearcherswhoatfirstfoundithardtobelieve.Choriocarcinomawasthefirstmalignanttumortobecuredbychemotherapy,and,mostremarkably,itcouldbecuredwithjustasingledrug,suchasmethotrexate.Chemotherapyworkedsowellagainstchoriocarcinoma,becausethecellsderivefromtheembryo,whichisaforeigntissue,asfarasthepatient’simmunesystemisconcerned.Aftermethotrexatekilledmosttherapidlydividingcancercells,theremainderwereoftenmoppedupbythepatient'simmunesystemreactingagainstthechoriocarcinomacellsthataregeneticallyderivedfromtheembryo.Theimmunesystemseesthiscancerasforeigntissue,becausemotherandchildarenotgeneticallyidentical:halfoftheembryo'sgenescomefromthefather.

Figure5.4.Choriocarcinoma,amalignanttumorthatmethotrexatecured.Itusuallyarisesintheplacentaofpregnantwomenandismadeupofwildlygrowingcellsof

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varioussizesandshapes.Some,suchasthelargedarkoneinnearthecenter,haveseveralnuclei.Thesearethecellsthatproducehumanchorionicgonadotropin(HCG)intheplacenta,aswellasinthetumor.WhentheHCGhormoneintheblooddeclinedtoundetectablelevels,itwasasignofresponseandeventualcure.Howmethotrexateworks--OverviewSkippingthedetailsfornow,theessentialpointisthatmethotrexateinhibitsthesynthesisofDNA:itpreventsthechromosomesfrombeingduplicatedforcelldivision.Inotherwords,itblocksthestepinthecelldivisioncyclewhereDNAhastobeduplicated.True,itisgoodtoblockthedivisionofleukemiaortumorcells,butnormalcellsinsomeimportanttissuesalsohavetoduplicateatahighrate,andinhibitingthosecellscancausemajorproblems.Thenormaltissuesmostsensitivetoblockageofcelldivisionbymethotrecatearetherapidlydividingblood-formingcellsinthebonemarrowandinthelining("mucosa")oftheintestines.Insomemalignancies,particularlyleukemias,tumorcellscanenterthebrain,wheremethotrexateiskeptoutbytheblood-brainbarrier.Thedrugwasthereforealsoinjectedintothecerebrospinalfluidbywayofaspinaltap,inordertokilltumorcellsthatmaybelurkinginthecentralnervoussystem(Whitesideetal.,1958).Folicacidisneededtoproducethechemicalbuilding-blocksrequiredtomakeDNA.Todoso,however,thefolicacidmoleculehastobealtered,firstbyadditionoftwohydrogenatomstoproducedihydrofolate,andthenadditionoftwomorehydrogenatomstoproducetetrahydrofolate.Thelatteristhereactionstepthatmethotrexateblocks(Figure5.5).Theenzymethatcarriesoutthisreactionisdihydrofolatereductase(DHFR),anditisthisenzymethatmethotrexatebindsandblocksMethotrexateusuallyhastobecombinedwithotheranticancerdrugstohavelastingbenefit.Thereisonetypeofcancerhoweverthatcanbecuredbymethotrexatealone,andthatischoriocarcinoma.Thisrarecancer,asalreadymentioned,occursduringpregnancyfromcellsintheplacentaoftheembryo.Thecellsofthisformofcancerdividerapidly,whichisonereasonthatthiscancerrespondssowelltoaDNAsynthesisinhibitorsuchasmethotrexate.Beforemethotrexate,metastaticchoriocarcinomawasfatalin90%ofcases(YarrisandHunter,2003).

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Figure5.5.Anessentialreactionthatmethotrexateinhibits.Thereactioniscarriedoutbytheenzymedihydrofolatereductase(DHFR),whichmethotrexatebindsandblocks.WhatDHFRdoesistoconvertthedouble-bondenclosedbythedashedoval(upperstructure)toasinglebond(lowerstructure).Thisinvolvesadding2hydrogens(notshown).Theproduct,tetrahydrofolate,isrequiredforthemanufactureofbuilding-blocksforDNA.Howmethotrexatekillscancercells.Methotrexate,likefolicacid,entersthecellbywayofchannelsthroughthecellsurfacemembrane.Cancercellsthathavetoofewofthosechannelsdon'tletmuchmethotrexateinandthereforedonotrespondwelltothedrug(Chenetal.,2013).Onceinsidethecell,anenzymeaddsseveralmoreglutamatestotheendofthemethotrexatemolecule,andthispolyglutamatedformcannotexitfromthecell,becausetheglutamatesbearnegativecharges,andelectricalchargeimpairstheabilityofmoleculestopassthroughthecellsurfacemembrane(Figure5.6).Moreover,molecularpumpsthatpumpmanydrugsoutofthecelldonotworkwiththepolyglutamatedformofmethotrexate(Chenetal.,2003).Thatwasimportantbecausedrugresistancewasoftencausedbyincreasedquantitiesofthosedrugeffluxpumps,butwouldthisresistancemechanismwouldnotworktoremovethepolyglutamatedmethotrexatefromthecell.Thusthepolyglytamateavoidedthiscommonresistancemechanism(Szaboetal.,2016).

Dihydrofolate-(FH2)-

Tetrahydrofolate-(FH4)-

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Asusual,however,thereal-lifesituationwasmorecomplicated:therewereenzymesinthecellthatremovedtheextraglutamates;folicacidcompeteswithmethotrexateforthepolyglutamatingenzyme;methotrexatepolyglutamatedrugscannotbegottendirectlyintothecell,becausetheywillnotpassthroughthecellmembrane(Szaboetal.,2016).Also,whenmethotrexatereducestheamountofthymidylateinthecell,afeedbacksignalinitiatesanattempttocompensatebymakingmoredihydrofolatereductaseenzyme(Rushworthetal.,2015).StrategieswerebeingdevelopedtoovercometheseproblemsMethotrexatethenbindsandinhibitsthekeytargetenzyme,dihydrofolatereductase(VolpatoandPelletier,2009).ThecellneedstheseenzymestoproducecomponentsrequiredforDNAsynthesis,particularlythymine,adenine,andguanine.Inalittlemoredetail,herearethestepsthatwerefoundtoberelevantfortheactionofmethotrexate:•Inthecell,folicacid(folate)readilypicksup2hydrogenatomstobecomedihydrofolate(FH2).•Dihydrofolatereductase(DHFR)thenadds2morehydrogenstoFH2toformtetrahydrofolate(FH4)(Figure5.5).MethotrexatebindstoandinhibitsDHFRandthereforeblocksthisreaction.Whathappensisthatthedrugbindstothefolatebindingsiteontheenzymeandpreventsnormalfolatefromcominginandbindingthere(VolpatoandPelletier,2009).•AnotherenzymeinthecelladdsamethylgrouptoFH4toformmethylene-tetrahydrofolate(meFH4),averyimportantmoleculethatmakesmethylgroupsavailableforthesynthesesofthymine,adenineandguanine.•meFH4providesamethylgroupfortheenzymethymidylatesynthasetomakethyminefromuracil(theenzymeconvertsdeoxyuridinephosphatetothymidinephosphate).Sincemethotrexateinhibitsdihydrofolatereductase,theproductionofFH4neededtomakemeFH4isblocked.WithoutmeFH4,thymidylatesynthasefunctionandtheproductionthyminecomponentsforDNAareimpaired.However,methotrexatealsoblocksthymidylatesynthasedirectly,whichmorecompletelyinhibitsthymidylateproduction.Result:byinhibitingdihydrofolatereductaseandthymidylatesynthase,methotrexateblockstheproductionofthymine,adenine,andguaninecomponentsforDNAsynthesis(Fangetal.,2016).

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Figure5.6.Methotrexatepolyglutamate.Threeglutamatesareshown,buttherecanbeasmanyas8.Notethenegativechargesonthe-CO2’s,whichpreventthemoleculefrompassingthroughthecell‘ssurfacemembrane.Whentheglutamatesareaddedinsidethecell,thepolyglutamatemethotrexatecannotfromthecell.HowcellsbecomeresistanttomethotrexateDrugresistance,eitherintrinsictothetumor,oracquiredthroughselectiveproliferationofresistantcells,isthemajorbugabooofchemotherapy.Resistancetomethotrexatewasfoundtobeduetoanyofseveralfactors;someofthebestunderstoodwerethefollowing(Walling,2006):•Methotrexateuptakechannelsthataretoofewinnumberorthathaveareducedbindingaffinityforthedrug;thedrugthencannotenterthecell(Sirotnaketal.,1968).•Reducedadditionofglutamatestothemethotrexatemolecule,therebyreducingtheretentionofthedruginthecell(Chenetal.,2003).•Increasedactivityoftheenzymethatremovestheextraglutamatesfrommethotrexatepolyglutamatesinsidethecell.Withouttheextraglutamates,thedrugcanescapefromthecell.•Reducedbindingaffinitybymutationofthedihyrofolatereductaseenzymeformethotrexate(VolpatoandPelletier,2009).Methotrexatewouldthenbeunabletoinhibittheenzyme.•Overproductionofdihydrofolatereductase(DHFR)byamplificationofthegene,i.e.,byanincreaseinthenumberofcopiesofthegeneinthecell'schromosomes(Flintoffetal.,1982).ThemethotrexatewouldthenbeunabletoblockalloftheincreasedamountofDHFRinsidethecell.Thisshowshowcomplextheproblemofovercomingdrugresistancecanbe.Mostofthechangescausingdrugresistancewereduetomutations,whichweremuchmore

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frequentincancercellsthaninnormalcells.Onlythemoreresistanttumorcellssurvived,butthesecouldkeepondividingtoformcancersthatdidnotrespondtothedrug.AmplificationoftheDHFRgeneinhomogeneouslystainingregions(HSR)ofchromosomes.Astrikingandunexpectedobservationwasmadein1976,byJuneBiedlerandBarbaraSpengleratMemorialSloan-KetteringCancerCenterinNewYork.Theywereexaminingthechromosomesofcellsthathadbeenmaderesistanttomethotrexateorotheranti-folatedrugs.Iimaginethatitmighthavebeenasurprise,orperhapsevenashock,toseethatamongthecell’schromosomestherewasonethatwasgreatlyelongated.Thereasonforthegreaterlengthappearedtobethatthechromosomehadaninsertionofalongregionthatwasdevoidoftheusualbandingpattern,aregionthattheythereforedubbed“homogeneouslystainingregion”(HSR)(Figure5.7).TheysurmisedcorrectlythattheHSRcontainedorwasmadeupofahugenumberofDHFRgenes–whichwasthecauseofthecell’sdrugresistance(BiedlerandSpengler,1976).ThestorywasconfirmedbyJackNunbergandcoworkersatColumbiaUniversityin1978(Figure5.8)(Nunbergetal.,1978).HSR’shavesincebeenfoundinchromosomesofmanycancers,generallyassociatedwithdrugresistanceattributabletoageneamplifiedintheHSR.

Figure5.7.Anexampleofahomogeneouslystainingregion(HSR)(arrow)inachromosomeofacancercell.AnHSRwaspresumedtobeanamplificationofagene,resultingindrugresistance(BiedlerandSpengler,1976).

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Figure5.8.AmplificationoftheDHFRgeneinahomogeneouslystainingregion(HSR)inachromosomeofamethotrexate-resistantChinesehamstercell.TheHSRcontainsahugenumberofDHFRgenes,whichgreatlyextendsthelengthofthechromosome.Thecorrespondingnormalchromosomeisontheright(Nunbergetal.,1978).Leucovorincomestotherescue.Chemotherapywithmethotrexateoftenrequiredhighdosagethatproducestroublingtoxicity,especiallytothebonemarrowandgastrointestinaltract.Fortunately,anantidotewasavailable:leucovorin(alsoknownasfolinicacidorcitrovorumfactor)(FlombaumandMeyers,1999;Schoenbachetal.,1950)(Figure5.9).Patientscouldtolerateupto50-foldhighermethotrexatedosesifleucovorinwasadministeredwithin24-48hours(Freietal.,1980).Thissocalled“high-dosemethotrexate/leucovorinrescue”regimengivenatweeklyintervalswasfoundeffective,especiallyincancersthatdonottakeupmethotrexatewell;thehighdosehelpstopushthedrugintothecells.Howmuchbetterthisregimenwasthanmethotrexatebyitself,however,remaineduncertain(Freietal.,1980;Zelceretal.,2008).MethotrexateinhibitedDNAsynthesisbyblockingbothdihydrofolatereductaseandthymidylatesynthase.Theseinhibitionscouldbereversedbyadministeringleucovorin(folinicacid,citrovorumfactor(Schoenbachetal.,1950).Therefore,whenhighdosesofmethotrexatewereneededforeffectiveanticancertreatment,leucovorinsuccessfullycounteredmethotrexate'smajortoxiceffectsonthebonemarrow,gastrointestinaltractandkidney,aswellastoxicitytothebrainandspinalcord,particularlyifthedrugwasadministeredintothespinalfluidtokillcancercellslurkinginthecentralnervoussystem(Whitesideetal.,1958).Leucovorinprovidedexcessdihydrofolate(FH2),whichcircumventedthemethotrexate-blockedreactionsteps(Howardetal.,2016).

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Figure5.9.Leucovorin(folinicacid,alsoknownascitrovorumfactor)isanaturalactivederivativeoffolicacid.Itdiffersfromfolicacidinhaving4hydrogensaddedtomakesingle-bonds(arrows)fromthedouble-bondsinfolicacid.ThereisaC=Oaddition(redoval),whichmakesamethylgroupavailableforthesynthesisofthymine,adenine,andguanine(inamannersimilartothecaseofmeFH4describedinChapter6).Platelettransfusiontocontrolbleedingbecomesessentialinthesearchforacure.Intheadvancedstagesofacuteleukemia,thenormalbonemarrowcellsbecomereplacedbyleukemiccells.Alife-threateningconsequenceisthatnotenoughplateletsaremadetocontrolbleeding,andthepatientisindangerofbleedingtodeath.Thatdangerlimitedtheamountofdrugthatcouldbesafelyadministered.Whentheproblemofhowtotransfusefreshplateletswassolved,theamountofdrugthatcouldbesafelyadministeredwasincreased.Platelettransfusionwasessentialforpatienttosurvivethedosageofthedrugcombinationsthatwereneededforcure.Hereishowplatelettransfusionbecamepossible:MuchofthecreditgoestoEmilJFreireich(“Jay”),whosepersonality,determination,andthinkingoutsidetheboxisentwinedinthestory.Freireich’sremarkablecareerandaccomplishmentswasdescribedinpoignantdetailbyJohnLaszlo(Laszlo,1995).FreireichcametotheNationalCancerInstituteshortlyaftertheNIHClinicalCenterwasopenedin1953.Sincehehadtrainedinhematology,GordonZubrodaskedhimtostartaLeukemiaprogram.EmilJFreireichimmediatelymetEmil(“Tom”)FreiIII,whodirectedtheNCI’sclinicalprogramandwhoseofficewasnextdoor.Theremarkablecoincidenceofthesimilarityoftheirnamescausedsomeconfusion.However,TomwaspreciseandsystematicincontrasttoJay’spredilectionfor“wild”

Leucovorin(Folinicacid)

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ideas,whichhepursuedrelentlessly,andwhichoftenworkedout.Theirnameswerealways“Tom”and“Jay”;theircommonname“Emil”wasneverused.Theycomplementedeachotherandtheircollaborationworkedextraordinarilywell.TheirdifferentpersonalitiesandwaysofthinkingwereveryevidentwhenIservedasaClinicalAssociateontheChildhoodLeukemiaServicein1957.TomFreiimpressedmeinthescopeofhisknowledge.Healwayshadastackofpunchedcardsinhislongwhitecoat,andansweredmyquestionswithreferencetopublishedevidence.JayhadsomeextraordinaryideathatIhaddifficultyaccepting;someofthemhoweverledtoimportantbreakthroughs,suchasthewayconcentratedbloodplateletscouldbestoredfortransfusion.Combinationchemotherapyincludingmethotrexatecureschildhoodleukemia.“Fullspeedaheadanddamnthetorpedoes.”Freireichwasfullof‘crazyideas’fornewtreatmentstotry.GordonZubrodasheadoftheMedicineBranchwasoftenskeptical,butneverthelessoftensupportedhim,becausetheoutlookforthechildrenwassobleak.Zubrod’sinstinctsborefruitasEmilJFreireich(“Jay”)wastodeservemuchofthecreditforthefirstcuresofchildhoodleukemia.ThedetailsofhowchildhoodleukemiawaseventuallycuredistoldinthebookbyJohnLazlo,whichalsodescribesthepersonalitieswhomadeitpossible(Laszlo,1995).Methotrexatewasakeypartofthedrugcombinationthatenabledthecures.Thestoriesoftheotheranti-cancerdrugsthatmadeupthefirstsuccessfulcombinationaretoldintheirrespectivechapters:vincristine,Chapter10;amethopterin(methotrexate),thischapter;6-mercaptopurine,Chapter7;prednisone,Chapter…ThetherapywasnamedVAMP,acombinationofthefirstlettersoftheaforementioneddrugnames.By1962,JayFreireichandTomFrei(EmilFrei)haddecidedthatitmadesensetocombinesomeofthedrugsthatindividuallyhadshownsomeactivity.Theyeventuallysoughttocombine4drugswithdifferentmechanismsofaction,butinadditionavoidingmultipledrugshavingthesametoxicity.Theideawastoattacktheleukemiccellsfromdifferentdirections,whileavoidingsynergistictoxicity.Theideaneverthelessstuckstrucksomecliniciansasfar-fetchedorcrazy(Laszlo,1995).Somethoughtthestudieswereneedlesslypoisoningtheverysickchildren.Nevertheless,JayFreireich’sdoggeddetermination,muchtothesurpriseofmany,curedsomeofthechildren.

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