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・UsingUGT1AsdoubleexpressionsystemsinHEK293ccllsthatpreviouslyestablishedinourlaboratory3'4),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.
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