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

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

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

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

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．

Refmmces：

1．YamanakaH,NakajimaM,HaraY;KatohM,nchibanaO,YamashitaJ,andYOkoiT

（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

（UGTm9*22)anditseffectsonthctranscriptionalactivity6P/ja7macoge"erics,14：

329-332.

-541-

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

UDP-glucuronosyltransferase2B21-catalyzedmorphine-6-glucuronidefOrmationMDノ

Ｐｈａ伽αＣＯ/,６０:1040-1048．

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

theconsequencesoftheY485Dmutationsontheactivityandsubstratｅａｆｆｉｎｉｔｙｏｆ

ＵＧＴ１Ａ６.Ｐ/iα肋acoge"ericsaMGe"o〃Cs,１7:１１ｓ-126．

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

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

ＵＧＴ１Ａ９,ａｎｄＵＧＴ２Ｂ１５:ｅｆｆｅｃｔｓｏｆＵＧ１[LUGTinteractions.、川gMCZZzbDiSpos,３ｓ：

1679-1686．

YamanakaH,NakajimaMKatohM,mdYOkoｉＴ(2007)G1ucuronidationofthyroxinein

humanliveLjCjunum,andkidneymicrosomes.、川9Ｍ℃m6Djqpos,35:164コ-1648.

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