3673

9000001143733

Grab, Dennis J.

Hirumi, Hiroyuki

English

OAK Obihiro University Archives of Knowledge

J．Pr【）【0ヱ00l．Rcs，，6，75－82（1ゲ始）  C叩γrfg九胞／99占．乃ど肋どdrC力Cビ〃′どrかP川‘卿α〃肋／gc〟J8rJm刑〟〃OJ♂gプ  rhnsferrinasa DrugCarrierinAf血amTiypanosomes  DENNISJ．GRABl′2ANDHIROYUKIHIRUMI3  旭甲血附呵〃血涙止別苅血吼勒函血Ⅵ血一館触ビdrC力Ce咄れC仰f嘲0〃・  山7肘ブj，U．∫．ん，2上）甲α伽〃rげ升甲血‖蹴fci腱，几Jα偲伽vどr咄5c力00Jげ舶J止肋d仙   β扇升叩血J加kd才r靴，〃ビルO′Je叫揖乃7J2，U・∫・Aりd扇3侮肋e8rぐんCe傭「摩r   pr∂J∂Z掘〃肋Jg（了“加血肌“〃0わgyO仙mU〃fver坤げAgr血血げgd扇陀Jピ′～朋り仙dic如  上J∽（ねc九0，伽摘Jr（），〃〃たhf血0β0，ノ甲γl  Receil・ed24Apri】1997／Accep【d5May1997   Keン・Wnrds＝叩q血；jゝ0－¶etamediumchlo血転Sa［nqrin；血－Sfenれ棚〟′冊閥e∂仰Jg〝∫g；丁仰d〃0∫p帽ムwどょ  ABSTRACT  We have coupled14cTSamorin to bovine transferrin（Tf）using2r  azidophenylglyoxal（APG），Samorin－APG－Tf wasincubatedwitheitherthe  TrypanosomacongolenseSamorin－SenSitive（ILl180）or Samorin－reSistant（IL  3338．2）The APG－Tfconjugatewithout bounddrug wasusedas thecontrol・  TrypanosomacongolenseIL1180grewpoorlyevenatSamorin／Tfconcentrations  aslowasO・12ngdrug／0・14ugTfperml・FreeSamorinkills77TPanOSOmL7IL  1180atadoseoflng／ml・Thee爪cacyofthedrug／Tfcomplexwasasleastas  effectiveasfreedrugaloneeveninthepresenceofhighamountsofserumTf  presentinmedium・ThesituationwiththeSamorinresistantT・COngOlenseclone  IL3338・2wasquitedifferent・ThiscloneissensitivetotheeffectsofSamorin－  APG－Tfatdrugconcentra（ionsoflOng／mlorhigher・SurprlSlngly，thecontroI  APG－TfconJugateWaSabletoinhibitthegrowthofbothTrwanosomalLl180  andIL3338．2，albeit at simi1arly highTfconcentrations：24・2－48・5鵬APG－  T〃ml．The data demonstratethe potentialuse ofTfasanove］drug delivery  systemin T・COngOlense・This finding has far reachingimplicatiomsinthe  treatmentofhumanSleeplngSicknessasTfisoneof（hefewserumproteinsthatis  abletocrosstheb10Od－brainbarrier．  INTRODUCTION  Africantrypanosomesareparasiticprotozoanflagellatesthatarefoundin   ・ ■   SAMORJN／TRANSFERRIN AND TRYPANOSOMES   thebloodofwildJife，】ivest∝kandman．Trypanosomesrequiretransferrin（Tf）  for growth（Schelleta）・1991a）andacquireitinareceptor－mediatedfhshion  （Coppensetal・1987；WebsterandGrab1988）．1ncontrasttomammaliancells，it  hasbeen shownthat Tfanditsreceptor are deliveredintothelysosomesof  African trypanOSOmeS and may not be recycled（Grab etal．1992，1993）．  COnSequently，Withtherightdrug／Tfcombination，itmayt光pOSSibletoconstruct  transftrrincomplexesthatwouldrecycleharmiesslyoutofmammalianCellsbut  betargeteddirectlylntOthelysosomesystemoftrypanosomestherebyprovidinga  degreeof spec摘citytothedelivery ofthedrugs totheirdesired target，the  trypanOSOme・Inthisreport，WedescribetheSynthesisofcomplexesofTfwiththe  antitrypanocidaldrugisometamediumchloride（Samorin－fromMay andBaker  Ltd，Dagenhan，England）using 2－aZidophenylglyoxalmonohydrate（APG：  Pierce）・Wealsodemonstrateinvitrothepotentialofthesecomplexesasnovel  drugdeliverysystemsinTrypanosomacongolense・This茄ndinghasfarreaching  implicationsin tIletreatmentOftrypanosomosis asTfis oneofthe fbw serum  proteins thatis able to crQSS the blood－brain barrier（Fishman etal．1987；  Pardridgeetal・1987；Shinetal．1995；Skarlotosetal．1995），amajorfactorin  thetreatmentofsleeplngSickness．   MÅrERIALSANDMEThOI）S  P花戸d相加〃q′∫α椚∂rf〃－APG－Ⅳco〝甲Jαe∫ご  Bovineapo－Tfwasloadedwithiron（KlausneretaJ．1983）andthediFe3＋－  TfseparatedfromfreeironbySephadexG－25chromatography・Crossslinkingof  SamorintoTfwasaccomplishedusingAPG，aheterobifunctionalphotoactivatable  CrOSS－1inkingreagentselectiveforarglnylresidues，aCCOrdingtothemethodof  Ngoetal．（1981）．Inthedark，0．8mgSamorincontainingtraceramountsof14c－  SanOrin［finalspecinc activity：2．5x105cpm／mg drug）in O．5ml100mM  NaHCO3（PH8．3 takeadvantageofthefactthatSamorinhasanarglnyl－1ikeresidueinthemolecule．  SamorinwasspecincallycrosslinkedtoSamorinbyovernightincubation（inthe  dark）atambienttemperature beforetheadditionlmldiFe3＋－Tf（50mg／mlin  buffer）．Transferrin was non－SpeCi鶴ca11y crosslinked to the APG－Samorin  COmplexbyexposuretobrighttroplCalsunlightfor10min．Aftercentrifugation  to remove any precIPitated material，the samples were passed twice through  SephadexG－25M（PharmaciaPD－10columns）equilibratedinphosphatebuffered  Saline（PBS）to remove unreacted drug and crosslinker．The speCinc activity，  basedonboundradioactivlty，OftheSamorin－APG－TfconJugateWaSeStimatedto  be531ngdrug／mgprotein，SuggeStlngthataboutlmoleculeinlOTfismodified．  Asacontrol，APGwasincubatedwithNaHCO3bufferwithoutdrugandcoupled  一「′－   SAMORIN／TRANSFERRIN AND TRYmNOSOMES   toTfexactlyasdescribedabove．Wealsospecjfica）1ycoupledAPGtoarBlnlnyl  residues on Tf before non－SpeCific（1ight reaction）addition of Samorin to the  APG－Tfcomplex．UndertheseconditionslmldiFe3＋－Tf（50mg／ml）inlOOmM  NaHCO3（pH8L3）wasfirstincubatedwith110ylAPG（10mg／mlDMSO）for36  hrsatroomtemperature（inthedark）followedbyadditionofO・5m18・3mM  Samorin．AfterexposuretosunlightthesamplewaspassedthroughSephadexG－  25Masabove．Thespecificactivity（basedonboundradioactivity）oftheTf－APG－  SamorinconJugateWaSeStimatedtobe429ngdrug／mgprotein，   血両肌＝血血油川q＝．co〃gOJ印∫e g〃血ク柁∫どれCg qr∫d∽∂rれAf〉G一群符」  APG－∫d肌∂rれⅣAPG一打…函押紙  TりpanOSOma COngOlense clonesILl180（Samorin sensitive）and T・  COngOlenseTL3338，2（Samorinresistant）bloodstreamformparasites（Hirumiand  Hirumi1993）were cu）tiva（ed jz124 wellcul（tlre Plates witJ）djfferent  concentrations of Samorin，APGLTf or APG－Tfin O．5 mlHMIL93 medium  （HirumiandHirumi199］）．HMト93medjumcol）SistsoflMDMsupplementedwith  20％younggoatserumand5％SerumPlus（HazletonBiologics，Lenexa，Kansas）  Whjchcontains）owlevels ofbovineserum（13yg／ml）aswellasbathocuproine  Sulphonate，L－CySteine，hypoxanthine，2－merCaptOethanol，PyruVateandthymidine・  Theparasites wereseededataconcentrationof4xlO5／ml・Ce11growthwas  SCreenedafter3－5daysaccordingtothesimplequalitativemeth（元ofHirumiand  Hirumi（1993）・lnthisassay system，tryPanOSOmeSthat grow welland reacha  plateauacidifythemediumwhichchangesthecoloroftheindicatordye，Pheno】  red，frompinktoyelLow（pHlessthan6．5）．Themediumremainspinkish（PH7・8－  8L5）incolorafterthe3－5dayincubationperiodwhentheparasitesarekiiledor  growpoorly・Theproteindeterminationwasperformedaccordingtothemethod  OfⅥncentandNadeau（1983）usingBSAasaproteinstandard．   REStJLTS  聯cJげ∫d椚OrJ和一APG－7r 7rAPG－∫d肌Orれ αJd APG一打c（叩〟g（‡fe∫ 0乃  仰α〃♂∫0椚ビg仰抄才力J〃γ∫J和．  SamorinrAPG－TfwasincubatedwitheithertheT．congolensestockILl180  0rJIJ3338・2・TheAPGrTfcoqugatewithoutbounddrugwasusedas［hecontrol・  As seenin Tablel，ILl180grew poorly even at thelowest Samorin／Tf  COnCentrationtested＝0・12ng Samorin／0・28vgTfperml．The resultsuslngTf－  APG－SamorlnWereSimi1ar・TrypanosomacongolenselLl180grewpoorlyinTf－  APGrSamorinwheretheconcentration ofSamorin／Tfwas greaterthanO・42ng  drug／0・79ygTf．FreeSamorinkills TLl180atadoseoflng／ml（H．Hirumi，  unpublished）．BloodstreamformsofIL2343were resistant to free Samorinat   SAMORINrTRANSFERRIN AND TRYfhNOSOMES  恥blelIncubationぱ物仰∫∂〝泌相関油乃∫どW仙S弧10rin－  APG－DIFe3十Iransferrin APG－Tr＊  Growthof7二c（，〃g（）～飢yg榊  TraTISfem11  Samorin  ］L3338．2  ILl180  138・00〝少雨  59，20咽凧  69．00  29．60  34．50  14．80  17．20  7．40   8．64  3．70   4．32  】．90   2．16  0．95  l．1ユ  0＿48   0．56  0．24   0．28  0．12   0．14  0．06   0．00  0．00  ＋ ＋正 ＊TheconeentrB（ionofTfaJldSamorinpresentinLheSamohn－   APG－Tfcomp】exesareglYenSeparately．  禅＊＋＝Jnhibitionofgrowth，－：Noinhibitionofgrowth  ¶血e2Incuba加n〔析物d伽∫〃〝追Cβ喝政舶βWithA和一DiFe3＋  tral】S托Ⅳi札  GⅢWtho†γぐ¢喝PJ仰e諷  APG－Tf＊   lL33粥．2  ILl柑0  194・0掴旭  97．0  48．5  24．2  ほ．1   0．0  ＋ ＋ 十＝†＋  ＊＋：lnhibitionDfgrowth，－ミNpinhib垣onofgrowth  ∴）   SAMORINrrRANSFERRIN AND TRYPANOSOMES   dosesofuptolng／ml（Hirumiand Hirumi1993）．Clearly，theefncacyofthe  drug／Tfcomplexwasanimprovemenfoverfreedruga】oneevenjnthepresence  OfhighaIT］OuntSOfnativegoatandbovinetransferrinpresentinHMI－93medium．  ThesituationwiththeSamorinresistantT，COngOlensecloneIL3338．2wasvery  different．This cloneis resistanttotheeffects ofSamorin atconcentrations oflO  ng／mlorhigher（HirumiandHirumi1993）．AsseeninTablel，theconcentration  Ofthedrug－APG－TfcoquBateWhichhadaneffectonthegrowthofT．congole72Se  IL3338．2contained14．8ng Samorin．Clearly，T．congoLenseIL3338．2isstill  resistant to Samorin，Unexpectedly，the controIAPG－Tfco町ugateWaS able to  inhibitthegrowthofbothT．coTZgOLenseILl180andIL3338．2simi1arly，albeitat  SimilarlyhighTfconcentrations：24．2－48．5mgAPG－Tf／ml（rIbble2）．   DISCUSSION  Ithasbeenshownthat，unlikemammaliancells，intheacidicenvironment  （pH4．2）ofthe trypanosome endosome／1ysosome system（Mbawaet al．1992）  Serum Tf，along withits receptor，in trypanosomesis delivered tolysosomes  （Grabetal・1992，1993；Sterverdingetal．1995）sugges【ingthattrypanosomes  haveevoIvedamodinedmechanismforironuptake．InT・bruceitheTfreceptor  has been shown to be an approximate］ylOO kDa protein（Grab et al・1993；  Sterverdingetal・1995）consistingofESAG6andESAG7（Schelletal・1991b；  Sterverdingetal・1995）・TheTfsysteminAfricantrypanosomesmaybeaunique  feature to these and possibly other Trypanosomatids・We have previously  PrOPOSedthatsinceTfisdeliveredtolysosomesinAfricantrypanosomes，itmay  bepossibletocovalentlybimddrugsandplantorbacterialtoxinstotransferrinfor  useasachemotherapeuticagent（Grabetal．1992）．Othershavedemonstratedthat  TfcanbeusedasatargetlngmOietyforrrlOleculesacrosstheblood－brainbarrier  PreSumably throughandinteractionwithTfreceptorsonbrainendothelialcdls  （Shinetal・1995），TTletryPanOCidaldrug，Samorin，isthoughttobetakeninto  trypanosomesviaatransportermoleculeintheplasmamembrar）e（Zilbersteinet  aJ・1993）・WehavebeenabletocoupleSamorintodiFe3＋－tranSferriTLuSingAPG，  aheterobifunctioTlalphotoafnnity crosslinking agentthat reactswitharglnlnyl  residues（Ngoetal．1981）．Samorin－APG－TfandAPG－Tfwereincubatedwith T．  COngOlenseparasitesknowntoberesistantorsensitivetoSamorin．Althoughthere  WaSnOChangeindrugseれSitivi【y5eenねr血eresista加7二ど∂〟g（）お那gC】皿e（IL  3338．2），theSamorinsensitiveT．congoLenseclone（IL1180）wasrenderedmore  SenSitive when drug was bound to Tf and probably delivered via the  endosoITle／1ysosomesystemratherthanatransporterroute（1もblel）．Clearlydrug  resistanceisdue to acombinationof events as suggestedby Kaminsky etal．  （1991）・Surprising）y，itwasalsofoLmdthatAPG－Tfcoq］Uga暮esalone，uSedasthe  7．ウ   SAMORIN／TRANSFERRIN AND TRYPANOSOMES   COntrOlinthestudy，Wereabletoinhibitthegrowthofbothresistantandsensitive  parasitest∝kstoasimilardegree，albeitatahighAPG－Tfconcentrations（Thble  2）・Wesuspect，buthavenotyetshown，thatAPG－derivitizedTfmayprecipitate  Outinthehydorphi11icenvironmentofthelysosoTne・  The possibilities of uslng Tf as a carrier for other drugS and other  molecu］essuchasotherdrugS，prOteaSeinhibitors，tOXins，anti－SenSeDNAand／or  antib∝ly（anti－TfR）combinations for use as potentia11yimportant anti－  trypanocidalagentsisaT”ttraCtiveone・However，in apreliminary study we  Observed thatSamorin－TfcomplexesdidれOtinhibitthein vitro growthofT．  bruceiwhichmayindicatethattheT・bruceEandT・COngOlenseclonesusedinour  StudiesintrinsicallyhandleTf／drugcomplexesdifferently・InthisrespeCt，Gerrits  et al・（1996）showed recently that T・bruceihave multiple expression sites  encoding for different mammal－SpeCi丘c Tf receptors which allow the  trypanosomesto efnciently takeupTffrom awide rangeofhosts・They also  bundthatbindingefncienciesfordifferentmammalianTfIscoulddifferasmuch  as100－hold・In our study，Samorin was coupled to bovine Tf．Since the T．  COngOZenseclonesusedinourstudywerealreadyadaptedtogrowinamedium  COntaining goat and bovine Tf，the parasites probably express Tf－reCeptOr  moleculeswhich binde餓cientlyto bovineTfLdrug・Ontheotherhand，theT．  bruceiclones we testedwere grown under axenicculture conditionsin Baltz■s  mod摘edminimumessentialmedium（Baltzetal．1985）asmodinedbyBlackand  l血nderveerd（1989）butcontaininglO％ratserum．Itjspossiblethatthebovine－  Samorin complexes were unable to compete effectively for rat－SpeCinc Tf－  receptorexpressedbytheparasite．   REFERENCI己S  Baltz，T．，Baltz，D．，Chiroud，C，H．，＆Crockett，）．1985．Cultivationin asemi－   de伽ed medium of animalinfective fbrms of TTyPanOSOma brucei，T．   叩痺W血肌r・飾∽尭T・rん0血∫血∫e弧d7’．g（‡沼地乃∫e．g〟βOJ．4ご1273－   1277．  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Transferrin as a Drug Carrier in African Trypanosomes

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Transferrin as a Drug Carrier in African Trypanosomes

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