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外因性化合物のグルクロン酸抱合の個体差に関する 研究

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of exogenous and endogenous compounds / 内因、

外因性化合物のグルクロン酸抱合の個体差に関する 研究

著者 Yamanaka Hiroyuki

著者別名 山中, 洋幸

雑誌名 博士学位論文要旨 論文内容の要旨および論文審査

結果の要旨/金沢大学大学院自然科学研究科

巻 平成20年6月

ページ 538‑543

発行年 2008‑06‑01

URL http://hdl.handle.net/2297/26880

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氏名 学位の種類 学位記番号 学位授与の日付 学位授与の要件 学位授与の題目

山中洋幸 博士(薬学)

博甲第1032号、

平成20年3月22日

課程博士(学位規則第4条第1項)

Intermdividualvariabilityofglucuromdationofexogenousandendogenous compounds.(内因、外因性化合物のグルクロン酸抱合の個体差に関する研究)

横井毅(医学系研究科・教授)

辻彰(自然科学研究科・教授),宮本謙一(医学部附属病院・教授),

加藤将夫(自然科学研究科・准教授),中島美紀(医学系研究科・准教授)

論文審査委員(主査)

論文審査委員(副査)

Abstract

G1ucuronidationwhichiscatalyzedbyUDP-glucuronosyltransferase(UGDenzymesisa majoreliminationrouteofmanyxenobioticsandendobiotics,andtherebyhasilnportantrolein efficacyandtoxicityofdrugaswellashonnonebalance・Thepurposeofmystudywasto investigatcthemtcrindividualvariabilityofglucuronidationof4,-HPPHandthyroxine,as examplcsofexogenousandendoge、oussubstratesofUGmrespectivelyFirst,invivo intcrindividualvariabilityin4,‐HPPHglucuronidationwasfOundtobellfO1dinl5patients、

ThevariabilitywasnotrelatedwithlmownpolymorphicmutationsofthcUGm4Z,UGrZda andUGTZaI9genes・BysequenceanalysesofUGZYIdgcneinapatient,anovelpolymoIpmc allele,tennedUGrM9*、,wasidcntifiedLuciferascassayrevealedthatthealleleincreases thetranscriptionalactivity6HowcveLtheallelewasnotrelatedwiththcvariabilityof4,‐HPPH glucuronidationinvivo・Second,invitrointerindividualvariabilityinthyroxine

glucuronidationwasfOundtobe4fO1dinl2humanlivermicrosomes・mwasdemonstratedthat intestmeandkidneywouldalsocontributetothethyroxineglucuronidationThird,itwas demonstratedthat4,‐HPPHandthyroxineglucuromdationscatalyzedbymultiplcUGT1A isofOnnswereaffectedbytheUGT-UGTinteractionnleinterindividualvariabilitywould resultfromthedifferencesintheexpressionlevelsofeacllUGTisofOnninliversamong individuals,extrahepaticglucuronidation,geneticpolymorphisms,andUGTUGTinteractions・

Thesefactorsmayintricatelyinteracteacllotllel;complicatingtheunderstaingingof interindividualvariabilityofglucuronidations.

Dissertationabstract

G1ucuronidationisamaJorrouteofeliminationofmanyxenobioticsandendobiotics,and therebyllasimportantroleinefficacyandtoxicityofdrugaswellashonnonebalance、

GlucuronidationiscatalyzedbyUDP-glucuronosyltransferases(UGrb).Thepurposeofmy

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endogenouscompounds・Theinterindividualvariabilityinglucuronidationsof4'一HPPHand thyroxmewasevaluatedasexogenousandendogenoussubstratesofUGmrespectively.

Interindividualvariamityofexo=enous4,-HPPH窪lucuronidation

Phenytoin,ananticonvulsantagent,ismainlyexcretedas4,-HPPHO-glucuronidcin humans・Phenytoinismetabolizedto41-HPPHbyCYP2C9andCYP2C19,and4,-HPPHis fiJrtherlnetabolizedto4,‐HPPHO-glucuronidebymultipleUGIもofUGT1A1,UGT1A4,

UGT1A6,andUGT1A9,4i-HPPHmaybebioactivatedtoareactivemetabolitethatis

associatedwitllsideeffectssuchasgingivalhyperplasia,somnolencc,drymonth,andgcncral fatigue、TherefOre,theglucuronidationisconsideredtobeadetoxificationpathwayJnthis studyltheextcntofintcrindividualvariabilityintheurinaryexcretionlevelsof4,-HPPHandits O-glucuronidcwasinrclatedwithgeneticpolymorphismsofUGZL4andCYP2C、4,-HPPH anditsglucuronideinurincsamplesfiDml5patientstowhomphenytoinwasadministcred weremcasuredbyliquidcllromatography-tandemmassspectrometry6Whenthemolarratioof 41-HPPHO-glucuronide/41-HPPHwascalculatedasanindexofglucuronidation,alargc interindividualvariability(11fO1d)wasobservedA1though5patientswerehcterozygotesof mutatedallelesofCYP2C9orCYP2CI9genes,norelationshipwithtlleinterindividual differenceinthetotalcxcretionof4i-HPPHanditsO-glucuronidewasobservedThe UGZMI*、UGn4I*2S,UGm4I*cOandUGm4G*ZalleleswerefOundinl,3,6,and8 paticnts,respectively・Therewasnorelationshipbetwecnthcgcneticpolymorphismsof

UGZYmsandthemtermdividualdifferenceinthe4'‐HPPHglucuronidation1).

nelucidatenewunlmownpolymorphicallclcofUG'IM11atmayaffectthe4'‐HPPH glucuronidation,allcxons,exon-intronjunctions,andthc5'一flankingregionoftheUGTMI,

UGTZ“UGm4aandUGTm9geneslnapatient,whohadextrelnelylow4'一HPPH

glucuronosyltransferaseactivityweresequenced・Astheresult,onebascinsertionofthymidinc inapromoterregionoftheUGTm9generesultinginA(1)1oArwasidcntifiedcomparingwith thcreferencescquenceofAFZ97093(A(T)9Ar).TheallelewastennedUGm49畑.nle luciferaseactivityofthepromoterconstmctcontainingthcA(r)1OATsequencewas26-fO1d higherthanthatoftheconstmctcontainingtheA(T)gATsequencc・Themutantallelewas expectedtoaltertheUGT1A9expressionleveLHowever,themutantallelewasnotassociated withthcinterindividualvariabilityin4,-HPPHglucuronidationinvivoinhuman2).Itwas reportcdthatcertainUGTisofOnnchangestheenzymeactivityofotherUGTisofOnns3'4,5).

Filrthennore,Kurkelactal6)recentlyrcportedthattheactivityofmutantUGTwasaffectedby

wildtypeofotherUGTisofCnns・TherefOre,invivoglucuronidationmaynotreflectthe cllangcdactivityoftheenzymebygeneticpolymorphismswhichwasevaluatedinvitro.

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4,-HPPHhasachiralccntcL⑥-4,-HPPHisaprcdommantfOnnproducedfTomphenytom mhumans,and(R)-4,-HPPHisancxtremclytoxiCfmmwithrespecttogingivalhypcrplasia、

Whenracemic4,-HPPHwasusedasasubstrate,humanlivcrmicrosomespredominantly

fOrmcd(8)-4,-HPPHO-glucuronidcratherthan(R)-4,-HPPHO-glucuronide・Amonghuman

UGTenzymcs,UGT1A1stereosclectivelyfCrmed(R)-4,-HPPHO-glucuronide,whcreas UGT1A9andUGTZB15stcreoselectivelyfDnned(9)-4,-HPPHO-glucuromdefromraccmic 4,-HPPH・UsingUGT1AsdoubleexpressionsystemsinHEK293ccllsthatpreviously

establishedinourlaboratory3'4),theeffectsofUGTUGTintcractiono、4,-HPPH

O-glucuronidefOnnationwereinvestigated,ItwasdemonstratedthatcoexpressionofUGT1A4 increasedtheVInaxvaluesof(3)-and(R)-4,-HPPHO-glucuronidefOnnationcatalyzedby UGT1A1,butdecreasedtheVmaxvaluesof(9)-and(R)-4,-HPPHO-glucuronidemnation catalyzedbyUGT1AQCocxpressionofUGT1A6increasedtheS50valuesanddccreasedthe Vmaxvalucsof(S)‐and(R)-4,-HPPHglucuronidefmnationcatalyzedbyUGT1A1and

UGT1A9、HoweveLtheinteractiondidnotalterthestereoselectivity7).hhumantissues,the

expressionlevelsofeacllUGTisofOrmandrelativeratioofthcmvaryamongindividuals,

indicatingthattheeffectsoftheUGTUGTinteractionswerediffCrentwitllintheindividuals,

Thus,theUGPUGTmtcractionwouldbeoneoffactorscausingtheinterindividualvariability ofglucuromdation.

lnterindividualvariabilityofendogenousthyroxineglucurOnidation

ThyroxineisamajorfOnnofthyroidhonnoncsecretcdfromthyroidgland,andisorally admmisteredinhypotllyroidismG1ucuronidationofthyroxineisamaJormetabolicpathway facilitatingitsexcrctionTheintcrindividualvariabilityofthyroxineglucuronidationmaybea casualfactoraffCctmgtheplasmathyroxineconccntrationhthisstudy,thcintcrindividual variabilityofthyroxincglucuronidationwereevaluated,andhumanUGTisofOImsinvolvedin theactivitywereidentificd・nrthennore,extrallepaticglucuronidationwcrecomparedwith hepaticglucuronidation・Eadic-HofSteeplotsofthyroxmeglucuronidationinllumanlive喝 jCjunum,andkidncymicrosomesweremonophasic、Humanjejunummicrosomesshoweda lowerKmvalue(24"M)thanhumanliver(8“M)andkidney(53浬M)microsomes・Human kidncymicrosomesshowedalowerVmaxvaluc(Z3pmol/in、/mg)thanhumanlivcr(l33 pmol/min/Ing)andjCjunum(185pmol/min/mg)microsomes・Byscaling-up,theinvivo clearancesinlivelうintestine,andkidneywereestimatedtobel440,702,and79’1/min/kgof bodyweigllt,respectively6TherefOre,thyroxineglucuronidationinextrahepatictissue,

especiallyinintestine,wouldcontributetothethyroxineglucuronidationinvivoUsing recombinanthumanUGTisofonnsexpressedinbaculovirus-infectedinsectcells,UGT1A8 (l09pmol/min/unit(S)),UGT1A3(gZpmol/min/unit(S)),andUGT1A10(47pmol/、血/imit

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glucuronosyltransferaseactMtymlcapparentKmvalueofrecombinantUGT1A1(105浬M)

wassimilartothatofhumanlivermicrosomes・Thethyroxineglucuronosyltransferaseactivity inmicrosomesfroml2humanliversrangedfromz3、7to848pmol/min/mgofprotein,

representmg4fO1dvariabilityltwassignificantlycorrelatedwithbilimbinO-(r=0.855,p〈

0.001)andestradiol3-O-C=0.827,p〈0.0001)glucuronosyltransferaseactivitiescatalyzed byUGT1A1,indicatmgthattheactivityinhumanliverismainlycatalyzcdbyUGT1AL KineticandinhibitionanalysessuggestedthatthethyroxineglucuronidationinhumanjCjunum microsomeswasmaimycatalyzedbyUGT1A8andUGT1A10andtoalesserextentUGT1A1,

andtheactivityinhumankidncymicrosomeswasmainlycatalyzedbyUGT1A7,UGT1Agand UGT1A10ThecontributionofeachUGT1AisofOrmwouldvarybetweenhumantissues,

dependingontherclativeabundanceofeachisofOrmWhcnthecffectsofUGTLUGT

interactiononthyroxineglucuronidesweremvestigated,itwasfOundthatthecoexprcssionof UGT1A4andUGT1A6differentlyaffCctedthekincticsofthethyroxincglucuronidcfOmation byUGT1A1orUGT1A9Thus,themteractionsmayaffectthcinterindividualvariabilityof

thyroxineglucuronidation8).

Conclusion

lnthisthcsis,theinterindividualvariabilityofglucuronidationofexogenousand

endogenouscompoudswascvaluated・Theinterindividualvariabilitywouldresultfromthe differencesintheexpressionlevelsofeachUGTisofOminliversamongindividuals,

extrallepaticglucuronidation,gcneticpolymorphisms,andUGTUGrinteractions・nlese factorsmayintricatclyinteracteachothe喝complicatingtheunderstandingofinterindividual variabilityofglucuronidations・Finally,theinfOnnationdescribedinthisthesiscouldfacilitate understandingabouttheintcrindividualvariabilitymglucuronidation.

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(2005)Urinaryexcretionofphenytoinmetabolites,

5-(4i-hydroxyphenyl)-5-phenylhydantoinanditsO-glucuronidemhumansandanalysisof geneticpolymolphismsofUDP-glucuronosyltransferases,D7zJgMbZzJbPノカα伽acokj"α,

2⑪:135-143.

ZYamanakaH,NakajimaM,KatohM,HaraY;machibanaO,YamashitaJ,McLcodHL,and YOkoiT(2004)AnovelpolymorphisminthepromoterregionofhumanWZYM9gene

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329-332.

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FUjiwaraR,NakajimaM,YamanakaH,NakamuraA,KatohMIkushiroS,Sakaki工and YokoiT(2007a)EffectsofcoexpressionofUGT1A9o、enzymaticactivitiesofhuman UGT1AisofOrms・DmgMb加わり⑰o3,35:747-757.

FmjiwaraR,NakajimaM,YamanakaH,KatohM,andYOkoiT(2007b)hteraction betweenhumanUGT1A1,UGT1A4,andUGT1A6affecttheirenzylnaticactivities・D7zJg

Mbm6DiWs,35:1781-1787.

IshiiY5MiyoshiA,WatanabeR,TbumdaK,耐udaM,Yamaguchi-NagamatsuY;YOshisue IUlanakaM,MajiD,OhgiyaS,andOguriK(2001)SimultaneousexpressionofgUinea

pigUDP-glucuronosyltransfCrase2B21and2B22inCOS-7cellsenhances

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KulkclaM,PatanaAS,MackenziePI,CourtMH,nteCGHiⅣonenJ,GoldmanA,and FinclM(2007)htcractionswithotherhumanUDP-glucuronosyltransferasesattenuate

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NakajimaM,YamanakaH,FUjiwaraR,KatohM,andYOkoiT(2007)Stereoselective

glucuromdationof5-(4i-hydroxyphenyl)-5-phenylhydantoinbyhumanUGT1A1,

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