༤ ኈ ㄽ ᩥ

In vitro ௦ㅰヨ㦂⣔࠾ࡼࡧࣄࢺ⫢⣽⬊࣓࣐࢟ࣛ࢘ࢫࢆ⏝࠸ࡓࣇ࢙ࣝࣂ࣓࣮ࢺࡢ཯ᛂᛶ௦ㅰ≀⏕ᡂ࣓࢝ࢽࢬ࣒ࡢゎᯒᗈᓥ኱Ꮫ኱Ꮫ㝔་ṑ⸆Ꮫ⥲ྜ◊✲⛉๰⏕་⛉Ꮫᑓᨷ
⏕యᶵ⬟ศᏊືែᏛ◊✲ᐊబ⸨ බஓ

┠ḟᗎㄽ................................................................................................................................ 1ᮏㄽ................................................................................................................................ 5➨ I ❶ ࣇ࢙ࣝࣂ࣓࣮ࢺ㸦FBM㸧ࡢ௦ㅰ࡟㛵୚ࡍࡿ⸆≀௦ㅰ㓝⣲ࡢ᳨ウ........................... 5➨ 1 ⠇ ⥴ゝ .................................................................................................................... 5➨ 2 ⠇ ᐇ㦂᪉ἲ ............................................................................................................. 8
I-2-i㸧 ໬ྜ≀ཬࡧヨ⸆ ............................................................................................. 8
I-2-ii㸧 ⫢࣑ࢡࣟࢯ࣮࣒ࢆ⏝࠸ࡓ FBM ࡢ௦ㅰヨ㦂 .................................................. 8
I-2-iii㸧 Cytochrome P450㸦CYP㸧ཬࡧ carboxylesterase㸦CES㸧Ⓨ⌧⣔࣑ࢡࣟࢯ࣮࣒
ࢆ⏝࠸ࡓ⸆≀௦ㅰ㓝⣲ࡢ᳨ウ....................................................................... 9I-2-iv㸧 ⤫ィゎᯒ ...................................................................................................... 9➨ 3 ⠇ ⤖ᯝ .................................................................................................................. 10
I-3-i㸧 ⫢࣑ࢡࣟࢯ࣮࣒ࢆ⏝࠸ࡓ FBM ࡢ௦ㅰヨ㦂 ................................................. 10
I-3-ii㸧 CYP ཬࡧ CES Ⓨ⌧⣔࣑ࢡࣟࢯ࣮࣒ࢆ⏝࠸ࡓ⸆≀௦ㅰ㓝⣲ࡢ᳨ウ ............. 15➨ 4 ⠇ ᑠᣓ .................................................................................................................. 17➨ II ❶ ࣄࢺ⫢⣽⬊࣓࣐࢟ࣛ࢘ࢫ㸦PXB ࣐࢘ࢫ㸧ࢆ⏝࠸ࡓ FBM ࡢ௦ㅰ≀ࣉࣟࣇ࢓࢖ࣝ
ཬࡧẘᛶホ౯ ..................................................................................................... 20➨ 1 ⠇ ⥴ゝ .................................................................................................................. 20➨ 2 ⠇ ᐇ㦂᪉ἲ ........................................................................................................... 24
II-2-i㸧 ໬ྜ≀ཬࡧヨ⸆.......................................................................................... 24
II-2-ii㸧 ᐇ㦂ື≀ ................................................................................................... 24
II-2-iii㸧 PXB ࣐࢘ࢫ࡟࠾ࡅࡿ FBM ࡢ௦ㅰ≀ࣉࣟࣇ࢓࢖ࣝホ౯............................ 24
II-2-iv㸧 ㉁㔞ศᯒ࢖࣓࣮ࢪࣥࢢἲ㸦MSI㸧ࢆ⏝࠸ࡓ glutathione㸦GSH㸧ࡢኚືゎᯒ................................................................................................................... 25
II-2-v㸧 FBM ࢆᢞ୚ᚋࡢ⫢ᶵ⬟ࣃ࣓࣮ࣛࢱ࣮ホ౯ ................................................ 26
II-2-vi㸧 ⤫ィゎᯒ................................................................................................... 26➨ 3 ⠇ ⤖ᯝ .................................................................................................................. 28
II-3-i㸧 PXB ࣐࢘ࢫ࡟࠾ࡅࡿ FBM ࡢ௦ㅰ≀ࣉࣟࣇ࢓࢖ࣝホ౯.............................. 28
II-3-ii㸧 MSI ࢆ⏝࠸ࡓ GSH ࡢኚືゎᯒ ................................................................. 36
II-3-iii㸧 FBM ࢆᢞ୚ᚋࡢ⫢ᶵ⬟ࣃ࣓࣮ࣛࢱ࣮ホ౯ ............................................... 39➨ 4 ⠇ ᑠᣓ .................................................................................................................. 40⤖ㄽ.............................................................................................................................. 44ㄽᩥ┠㘓 ............................................................................................................................ 46iㅰ㎡.............................................................................................................................. 47ཧ⪃ᩥ⊩ ............................................................................................................................ 48

ii

␎ㄒ୍ぴ
ᮏㄽᩥ࡟࠾࠸࡚ࡣ௨ୗࡢ␎ㄒࢆ⏝࠸ࡓࠋ

2OH-FBM:2-hydroxy felbamate

2-PP:

2-phenylpropenal

ALP:

alkaline phosphatase

ALT:

alanine aminotransferase

AST:

aspartate aminotransferase

CBMA:

3-carbamoyl-2-phenylpropionaldehyde

CCMF:

4-hydroxy-5-phenyltetrahydro-1,3-oxazin-2-one

CES:

carboxylesterase

CPPA:

3-carbamoyl-2-phenylpropionic acid

CYP:

cytochrome P450

ESI:electrospray ionization

FBM:

felbamate

FDA:

US Food and Drug Administration

GSH:

glutathione (reduced form)

GSSG:

glutathione (oxidized form)

HPLC:

high-performance liquid chromatography

ICH:

Interational Council for Harmonisation of Technical Requirements for
Pharmaceuticals for Human Use

IDT:

idiosyncratic drug toxicity

LC-MS:

liquid chromatography-tandem mass spectrometry

LC-UV/MS:

liquid chromatography-tandem mass spectrometry coupled with UV detector

MALDI:

matrix assisted laser desorption/ionization

MCF:

monocarbamate felbamate

MCF-gluc:

glucuronide of MCF[M + H]+:

proton adduct[M + K]+:

potassium ion adduct

MSI:

mass spectrometry imaging

MW:molecular weightiii

MW175:felbamate metabolite with molecular weight of 175

MW295:

felbamate metabolite with molecular weight of 295

MW439:

felbamate metabolite with molecular weight of 439

MW441:

felbamate metabolite with molecular weight of 441

MW455:

felbamate metabolite with molecular weight of 455

m/z:

mass-to-charge ratioNac-acid:

N-acethylcystein conjugated metabolite of 2-PP

Nac-OH:N-acethylcystein conjugated metabolite of 2-PP

NADPH:

nicotinamide adenine dinucleotide phosphate

PXB-mice:chimeric mice with humanized liver

pOH-FBM:p-hydroxy felbamate

SCID:

severe combined immunodeficiency disease

SULT:

sulfotransferaseSD:standard deviation

uPA:

urokinase-type plasminogen activator

UGT:

uridine 5’-diphospho-glucuronosyl transferase

UV:

ultravioletivᗎㄽ๰⸆◊✲࡟࠾ࡅࡿࢥࣥࣅࢼࢺࣜ࢔ࣝࢣ࣑ࢫࢺ࣮ࣜࡢᑟධࡸ㸪໬ྜ≀ࣛ࢖ࣈ࣮ࣛࣜࡢᣑ඘㸪ࣁ࢖ࢫ࣮ࣝࣉࢵࢺ࡞ホ౯⣔ࡢᵓ⠏࡞࡝࡟ࡼࡾ㸪௒᪥㸪ᩘከࡃࡢ໬ྜ≀ࡀ་⸆ရೃ⿵࡜ࡋ࡚ホ౯ࡉࢀ࡚࠸ࡿࠋ๰⸆ẁ㝵࡛ࡣ 1 ୓௨ୖࡢ໬ྜ≀ࡀࢫࢡ࣮ࣜࢽࣥࢢ࡟ࡼࡾホ౯ࡉࢀ㸪ື≀ᐇ㦂࡞࡝࡟ࡼࡿ⤠ࡾ㎸ࡳࡀ⾜ࢃࢀࡓࡢࡕ㸪⮫ᗋヨ㦂ࢆ⤒࡚ࡼ࠺ࡸࡃ 1 ࡘࡢ་⸆ရࡀୡࡢ୰
࡟⏕ࡳฟࡉࢀࡿࠋࡇ࠺ࡋࡓ་⸆ရࡢ◊✲㛤Ⓨ㐣⛬࡟࠾࠸࡚㸪⮫ᗋヨ㦂ẁ㝵࡟ධࡗ࡚㛤Ⓨࢆ୰Ṇࡍࡿࡇ࡜ࡣ㸪ࢥࢫࢺ㠃ࡢ㈇ᢸࡸ᫬㛫ࡢࣟࢫࡀ኱ࡁࡃ㸪⮫ᗋヨ㦂ࡢᡂຌ☜⋡ࢆୖࡆࡿࡇ࡜ࡣ〇⸆఍♫ࡢࡳ࡞ࡽࡎ㸪᪂⸆ࢆᚅࡕᮃࡴᝈ⪅࡟࡜ࡗ࡚ࡶ㔜せ࡛࠶ࡿࠋ་⸆ရࡢ㛤Ⓨ୰Ṇࡢཎᅉ࡜ࡋ࡚㸪1990 ᖺ௦๓༙㡭ࡲ࡛ࡣ⸆≀ືែࡀ୺࡞ཎᅉ࡜ࡋ࡚ᣲࡆ
ࡽࢀ࡚࠸ࡓࡀ㸪ࡑࡢᚋ㸪ࣄࢺ⤌⧊ࢆ฼⏝ࡋࡓ⸆≀ືែ◊✲ࡀྍ⬟࡜࡞ࡗࡓࡇ࡜࡞࡝࡟ࡼࡾ㸪
2000 ᖺ௦࡟ࡣ⸆≀ືែࡀཎᅉ࡛ࡢ་⸆ရࡢ㛤Ⓨ୰Ṇࡣ኱ᖜ࡟ῶᑡࡋࡓ㸦Kola et al.㸪2004㸧ࠋ୍᪉࡛㸪⸆ຠࡸẘᛶ㸦๪స⏝㸧ࡀཎᅉ࡛㛤Ⓨ୰Ṇ࡜࡞ࡗࡓ໬ྜ≀ࡢ୰࡟ࡶ㸪ࡑࡢせᅉ࡜ࡋ࡚⸆≀ືែࡀ㛵ಀࡋ࡚࠸ࡿ౛ࡀከ࠸ࡇ࡜ࡀᣦ᦬ࡉࢀ࡚࠾ࡾ㸦Kola et al.㸪2004㸧㸪⸆≀ືែࡢ
どⅬ࠿ࡽ⸆ຠࡸẘᛶࢆ⪃ᐹࡍࡿࡇ࡜ࡣ㔜せ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋ

་⸆ရࡢ⸆ຠࡸẘᛶࣜࢫࢡࢆ⸆≀ືែ㠃࠿ࡽ⪃ᐹࡍࡿୖ࡛ࡣ㸪ᮍኚ໬య࡟ຍ࠼࡚ࣄࢺ࡛
⏕ᡂࡉࢀࡿ௦ㅰ≀ࢆணࡋ㸪ホ౯ࡍࡿࡇ࡜ࡀ㔜せ࡛࠶ࡿࠋ་⸆ရࡢ◊✲㛤Ⓨ㐣⛬࡟࠾ࡅࡿ௦ㅰ≀ホ౯࡟ࡘ࠸࡚ࡣ㸪2008 ᖺ࡟⡿ᅜ㣗ရ་⸆ရᒁ㸦US Food and Drug Administration㸹FDA㸧
࡟ࡼࡾⓎ⾜ࡉࢀࡓࠕMetabolites in Safety Testing㸦MIST㸧ࠖ࢞࢖ࢲࣥࢫཬࡧ 2009 ᖺ࡟᪥⡿ EU
་⸆ရつไㄪ࿴ᅜ㝿఍㆟㸦Interational Council for Harmonisation of Technical Requirements for
Pharmaceuticals for Human Use㸹ICH㸧ࡼࡾⓎ⾜ࡉࢀࡓࠕM3(R2) Nonclinical Safety Studies for
the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals㸦 ICH
M3(R2)㸧
ࠖ࢞࢖ࢻࣛ࢖࡛ࣥᐃࡵࡽࢀ࡚࠸ࡿࠋ
ࡇࢀࡽࡢつᐃ࡛ࡣ㸪ᮍኚ໬య࠶ࡿ࠸ࡣ⸆≀㛵㐃≀㉁ࡢ⥲᭚㟢㔞࡟ᑐࡋ࡚ 10%ࢆ㉸࠼ࡿ௦ㅰ≀࡟ࡘ࠸࡚ࡣ㸪ẘᛶヨ㦂࡟࠾ࡅࡿ௦ㅰ≀ࡢ᭚㟢㔞ࢆ⪃៖ࡋࡓୖ࡛㸪㠀⮫ᗋヨ㦂࡛௦ㅰ≀ࡢẘᛶホ౯ࢆࡍࡿࡇ࡜ࡀᐃࡵࡽࢀ࡚࠾ࡾ㸪ࣄࢺࡢ⸆≀௦ㅰࣉࣟࣇ࢓࢖ࣝ࡟ᇶ࠸ࡓẘᛶࣜࢫࢡホ౯ࡣ㸪ࡼࡾᏳ඲࡞་⸆ရࢆᥦ౪ࡍࡿࡇ࡜࡟ຍ࠼㸪㛤Ⓨࢥࢫࢺࡢపῶ㸪㛤Ⓨᮇ㛫ࡢ▷⦰࡟࠾࠸࡚ࡶ㔜せ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋ
1ࣇ࢙ࣝࣂ࣓࣮ࢺ㸦felbamate㸹FBM㸧ࡣ 1993 ᖺ࡟ FDA ࡟ࡼࡾᢎㄆࡉࢀࡓᢠ࡚ࢇ࠿ࢇ⸆࡛࠶ ࡾ 㸪 ໭ ⡿ ࡟ ࠾ ࠸ ࡚ Felbatol® ࡢ ၟ ရ ྡ ࡛ Ⓨ ኎ ࡉ ࢀ ࡚ ࠸ ࡿ 㸦 United States Prescribing
Information: felbatol®㸧ࠋFBM ࡢᵓ㐀ࢆ Fig. 1 ࡟♧ࡍࠋFBM ࡣᐇ㦂ື≀ࢆ⏝࠸ࡓ㠀⮫ᗋヨ㦂ཬࡧࣄࢺ࡟࠾ࡅࡿ⮫ᗋヨ㦂࡛ࡣ㢧ⴭ࡞ẘᛶࢆ♧ࡉ࡞࠿ࡗࡓ୍᪉㸪ᕷ㈍ᚋ࡟ FBM ࡢᢞ୚ࢆཷࡅࡓᝈ⪅࡟࠾࠸࡚㸪㔜⠜࡞⫢ẘᛶཬࡧ㦵㧊ẘᛶࡀⓎ⏕ࡋ㸦Kaufman et al.㸪1997㸧
㸪㯮ᯟ㆙࿌㸦black box warning㸧ࡀබᕸࡉࢀࡿࡇ࡜࡟࡞ࡗࡓࠋࡇࢀ࡟ࡼࡾ㸪⌧ᅾ㸪FBM ࡢ౑⏝ࡣ㞴἞ᛶࡢ࡚ࢇ࠿ࢇᝈ⪅࡟㝈ᐃࡋ㸪ࣜࢫࢡ࡜࣋ࢿࣇ࢕ࢵࢺࢆ຺᱌ࡋ࡚ៅ㔜࡟ᢞ୚ࡍࡿࡇ࡜ࡀồࡵࡽࢀ࡚࠸ࡿ㸦United States Prescribing Information: felbatol®㸧ࠋFig. 1. Chemical structure of felbamate.

⸆≀࡟ࡼࡿẘᛶࡣ㸪ࠕ୰ẘᛶࠖ࡜ࠕ≉␗య㉁ᛶࠖࡢ 2 ࡘ࡟኱ูࡉࢀࡿࠋ୰ẘᛶ⸆≀ẘᛶࡣ㸪⏝㔞౫Ꮡᛶࡀ࠶ࡾື≀ᐇ㦂ࡢ⤖ᯝࡀࣄࢺ࡟እᤄྍ⬟࡛࠶ࡿࠋ୍᪉㸪≉␗య㉁ᛶ⸆≀ẘᛶ
㸦idiosyncratic drug toxicity; IDT㸧ࡣ㸪ẘᛶࡢⓎ⌧࡟⏝㔞౫Ꮡᛶࡀ↓ࡃ㸪ࡲࡓ㸪Ⓨ⑕㢖ᗘࡀ⛥࡛࠶ࡿࡓࡵ࡟ື≀ᐇ㦂ࡸ⮫ᗋヨ㦂࡛ࡣ᳨ฟࡍࡿࡇ࡜ࡀᅔ㞴࡛㸪་⸆ရ࡜ࡋ࡚ᢎㄆࡉࢀ㸪ࡼࡾከࡃࡢᝈ⪅࡟ᢞ୚ࡉࢀࡿࡼ࠺࡟࡞ࡗ࡚ึࡵ᳨࡚ฟࡉࢀࡿ㸦Ikeda㸪2015㸧
ࠋIDT ࡟ࡼࡿẘᛶ
Ⓨ⌧࣓࢝ࢽࢬ࣒࡟㛵ࡋ࡚ࡣ୙᫂࡞Ⅼࡀከ࠸ࡶࡢࡢ㸪ᑡ࡞ࡃ࡜ࡶ௨ୗ࡟♧ࡍ 3 ࡘࡢせᅉࡀ㛵୚ࡋ࡚࠸ࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋձ ⸆≀せᅉ㸸཯ᛂᛶ௦ㅰ≀࡜ࡼࡤࢀࡿ཯ᛂᛶࡢ㧗࠸௦ㅰ≀ࢆయෆ࡛⏕ᡂࡍࡿࡇ࡜࡟⏤᮶ࠋղ 㑇ఏⓗせᅉ㸸⸆≀௦ㅰ㓝⣲ࡸࢺࣛࣥࢫ࣏࣮ࢱ࣮㸪ཷᐜయ࡞࡝ࡢ㑇ఏᏊከᆺ࡟⏤᮶ࠋճ ⎔ቃせᅉ㸸㣗⏕άࡸ⫧‶㸪㣧㓇㸪ႚ↮࡞࡝ࡢ⏕ά⩦័࡟⏤᮶ࠋ
2

ࡇࡢ 3 ⪅ࡀ」ྜⓗ࡟㛵୚ࡋ࡚Ⓨ⑕ࡍࡿࡶࡢ࡜⪃࠼ࡽࢀ࡚࠸ࡿ㸦ᒣ⏣ࡽ㸪2006㸧
㸦Fig. 2㸧ࠋࡑࡢ୰࡛ࡶ≉࡟཯ᛂᛶ௦ㅰ≀ࡢ⏕ᡂࡣ IDT ࡢⓎ⌧࡟㛵୚ࡍࡿ㔜せ࡞ᅉᏊ࡛࠶ࡿ࡜⪃࠼ࡽࢀ
࡚࠾ࡾ㸦Park et al.㸪1998㸧㸪་⸆ရࡢ◊✲㛤Ⓨ࡟࠾࠸࡚ࡣ㸪཯ᛂᛶ௦ㅰ≀ࡢࣄࢺ⫢⣽⬊࡬ࡢඹ᭷⤖ྜ㔞ࢆᣦᶆ࡟ẘᛶࣜࢫࢡࢆホ౯ࡍࡿᡭἲࡀྲྀࡾධࢀࡽࢀ࡚࠸ࡿ㸦Nakayama et al.㸪2009㸧ࠋDrug factors
Reactive metabolite(Chemical structure, Dose,
PK, etc.)

Lifestyle(Food, Obesity,
Alcohol, Smoking, etc.)

Genetic polymorphism(Enzyme, Receptor,
Transporter, etc.)

Genetic factors

Environment factors
Fig. 2. Three factors of IDT

IDT ࡟ࡼࡿ⫢ẘᛶࢆⓎ⌧ࡍࡿ⸆๣ࡢ౛࡜ࡋ࡚㸪྾ධ㯞㓉⸆࡛࠶ࡿ halothane ࡀᣲࡆࡽࢀࡿࠋHalothane ࡣ㸪10000 ே㹼100000 ே࡟ 1 ே࡜࠸࠺㢖ᗘ࡛࢔ࣞࣝࢠ࣮ᛶ⫢㞀ᐖࢆⓎ⑕ࡍࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿࡀ㸦Park et al.㸪2005㸧㸪ࡇࡢ⫢㞀ᐖࡣ㸪⫢⮚࡛ cytochrome P450㸦CYP㸧
2E1 ࡟ࡼࡿ௦ㅰⓗάᛶ໬ࢆཷࡅ࡚⏕ᡂࡍࡿ཯ᛂᛶ௦ㅰ≀ trifluoroacetyl chloride ࡀ㸪ࢱࣥ
ࣃࢡ㉁ࢆࢺࣜࣇࣝ࢜ࣟ࢔ࢭࢳࣝ໬ࡋ࡚ᢠཎᛶࢆ♧ࡍࡇ࡜࡟ࡼࡾᘬࡁ㉳ࡇࡉࢀࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿ㸦Park et al.㸪2005㸹Satoh et al.㸪1985㸹Walgren et al.㸪2005㸧ࠋࡲࡓ㸪ୡ⏺ึࡢ࢖ࣥࢫࣜࣥ᢬ᢠᛶ⢾ᒀ⑓἞⒪⸆࡜ࡋ࡚ᢎㄆࡉࢀࡓ troglitazone ࡣ㸪Ṛஸ౛ࢆྵࡴ㔜⠜࡞⫢㞀ᐖࡢⓎ⏕࡟ࡼࡾ㸪Ⓨ኎࠿ࡽࢃࡎ࠿ 3 ᖺ࡛ᕷሙ᧔㏥ࡍࡿࡇ࡜࡜࡞ࡗࡓ㸦Walgren et al.㸪2005㸧ࠋTroglitazone ࡟ࡼࡿ⫢ẘᛶ࡟ࡣ㸪࣐ࢡࣟࣥ⎔⏤᮶ࡢ࢟ࣀ࢚࣏ࣥ࢟ࢩࢻࡸ࢟ࣀ࣓ࣥࢳࢻᆺࡢ
཯ᛂᛶ௦ㅰ≀㸪࠶ࡿ࠸ࡣࢳ࢔ࢰࣜࢪࣥࢪ࢜ࣥ⎔⏤᮶ࡢࢫࣝࣇ࢙ࣥ㓟ࡸࢣࢺ࢖ࢯࢩ࢔ࢿ࣮
ࢺᆺࡢ཯ᛂᛶ௦ㅰ≀ࡀ㛵୚ࡍࡿࡇ࡜ࡀ♧၀ࡉࢀ࡚࠸ࡿ㸦Kassahun et al.㸪2001㸹Smith㸪2003㸹
Tettey et al.㸪2001㸧
ࠋ

3

FBM ࡟ࡼࡿ⫢ẘᛶࡶ㸪⸆≀ࡢ௦ㅰ⮚ჾ࡛࠶ࡿ⫢⮚࡟࠾࠸࡚㸪཯ᛂᛶ௦ㅰ≀ࢆ⏕ᡂࡍࡿࡇ࡜࡟ࡼࡾⓎ⏕ࡍࡿ IDT ࡛࠶ࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿࡶࡢࡢ㸪FBM ࡢ௦ㅰ࡟㛵୚ࡍࡿ㓝⣲ࢆྵࡵ㸪ࡑࡢヲ⣽ࡣ᫂ࡽ࠿࡟࡞ࡗ࡚࠸࡞࠸ࠋࡑࡇ࡛ᮏ◊✲࡛ࡣ㸪FBM ࡢ཯ᛂᛶ௦ㅰ≀⏕ᡂ࣓࢝ࢽࢬ࣒ࡢゎ᫂ཬࡧࡼࡾᏳ඲࡞ FBM ࡢ⮫ᗋ౑⏝࡬ࡢ㈉⊩ࢆ┠ⓗ࡜ࡋ᳨࡚ウࢆᐇ᪋ࡋ
ࡓࠋ

4

ᮏㄽ
➨ I ❶ FBM ࡢ௦ㅰ࡟㛵୚ࡍࡿ⸆≀௦ㅰ㓝⣲ࡢ᳨ウ➨ 1 ⠇ ⥴ゝ

FBM ࡢ⮫ᗋヨ㦂࡛ࡣ㸪[14C]ᶆ㆑ FBM㸦[14C]FBM㸧ࢆ⏝࠸ࡓ࣐ࢫࣂࣛࣥࢫヨ㦂࡟࠾࠸࡚㸪
FBM ࡢ௦ㅰ≀࡜ࡋ࡚㸪FBM ࡢ㓟໬௦ㅰ࡟ࡼࡾ⏕ᡂࡍࡿ 2-hydroxy felbamate㸦2OH-FBM㸧ཬࡧ p-hydroxy felbamate 㸦 pOH-FBM 㸧㸪 ࡲ ࡓ FBM ࡢ ຍ Ỉ ศ ゎ ௦ ㅰ ࡟ ࡼ ࡾ ⏕ ᡂ ࡍ ࡿ
monocarbamate felbamate㸦MCF㸧࡜㸪MCF ࡀ᭦࡟㓟໬௦ㅰࢆཷࡅ࡚⏕ᡂࡍࡿ 3-carbamoyl-2phenylpropionic acid㸦CPPA㸧ࡢ 4 ࡘࡢ௦ㅰ≀ࡀྠᐃࡉࢀࡓ㸦Adusumalli et at.㸪1993㸹Yang etal.㸪1991㸧ࠋᕷ㈍ᚋ࡟ FBM ࢆᢞ୚ࡋࡓᝈ⪅࡟࠾࠸࡚㔜⠜࡞⫢ẘᛶࡀⓎ⏕ࡋࡓࡇ࡜࠿ࡽ㸪ᮍྠᐃࡢ୰㛫௦ㅰ≀ࢆྵࡵࡓ FBM ࡢ௦ㅰ≀࡟㛵ࡍࡿ◊✲ࡀ⾜ࢃࢀ㸪ࡑࡢ୰࡛㸪MCF ࠿ࡽ
CPPA ࢆ⏕ᡂࡍࡿࡲ࡛ࡢ㛫࡟୰㛫௦ㅰ≀࡜ࡋ࡚࢔ࣝࢹࣄࢻᆺࡢ௦ㅰ≀ 3-carbamoyl-2phenylpropionaldehyde㸦CBMA㸧ࡀᏑᅾࡍࡿྍ⬟ᛶࡀᥦၐࡉࢀࡓ㸦Thompson et al.㸪1996㸧
ࠋ
CBMA ࡀ⏕ᡂࡍࡿ࡜㸪ȕ 㞳⬺཯ᛂ࡟ࡼࡾ㸪Į㸪ȕ ୙㣬࿴࢔ࣝࢹࣄࢻ࡛࠶ࡿ 2-phenylpropenal
㸦2-PP㸧ࡀ⏕ᡂࡍࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿ㸦Thompson et al.㸪1996㸧ࠋĮ㸪ȕ ୙㣬࿴࢔ࣝࢹࣄࢻࡣ㸪ᢠᝏᛶ⭘⒆⸆ cyclophosphamide ࡢ௦ㅰ≀࡛࠶ࡿ acrolein ࡟௦⾲ࡉࢀࡿࡼ࠺࡟㸪୍⯡ⓗ࡟㠀ᖖ࡟㧗࠸ồ㟁Ꮚᛶࢆ♧ࡍࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ㸦Dieckhaus et al.㸪
2002㸧ࠋThompson ࡽ㸦1996㸧ࡣ㸪FBM ཬࡧ 2-PP ࢆྵࡴ」ᩘࡢ FBM ௦ㅰ≀ࢆྜᡂࡋ㸪in vitro࡛⣽⬊㞀ᐖάᛶࢆ᳨ウࡋࡓ࡜ࡇࢁ㸪2-PP ࡢ⣽⬊㞀ᐖάᛶࡀࡶࡗ࡜ࡶ㧗ࡃ㸪2-PP ࡢ⣽⬊㞀ᐖάᛶࡣ㸪Į㸪ȕ ୙㣬࿴࢔ࣝࢹࣄࢻ࡛࠶ࡿ acrolein ࡸ 4-hydroxynonenal ࡜ẚ㍑ࡋ࡚ࡶ᭱ࡶ㧗࠿ࡗࡓࡇࢆሗ࿌ࡋ࡚࠸ࡿࠋࡲࡓ㸪2-PP ࡣࣜࣥ㓟⦆⾪ᾮ୰࡛ glutathione㸦GSH㸧࡜ᐜ᫆࡟཯ᛂ
ࡋ㸪GSH ࡜ࡢ⤖ྜయࢆ⏕ᡂࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ㸦Dieckhaus et al.㸪2001㸧ࠋࡇ࠺ࡋࡓ
᝟ሗࢆ㋃ࡲ࠼㸪⌧ᅾ㸪FBM ࡟ࡼࡿ⫢ẘᛶࡢⓎ⌧࡟ࡣ཯ᛂᛶ௦ㅰ≀࡜ࡋ࡚ 2-PP ࡀ㛵୚ࡋ࡚࠸ࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋ

5

[14C]FBM ࢆ⏝࠸ࡓ࣐ࢫࣂࣛࣥࢫヨ㦂࡟ຍ࠼㸪ࡑࡢᚋࡢ in vitro ヨ㦂ࡢ⤖ᯝࡶ㋃ࡲ࠼࡚㸪⌧ᅾ㸪FBM ࡢ௦ㅰ⤒㊰࡜ࡋ࡚ࡣ Fig. 3 ࡟♧ࡍ௦ㅰ⤒㊰ࡀ᥎ᐃࡉࢀ࡚࠸ࡿࠋ

FBM ࡣ➨୍ẁ㝵┠ࡢ௦ㅰ࡜ࡋ࡚㸪㓟໬࠶ࡿ࠸ࡣຍỈศゎࢆཷࡅ㸪㓟໬௦ㅰ࡟ࡼࡾ 2OHFBM ཬࡧ pOH-FBM ࢆ⏕ᡂࡋ㸪ຍỈศゎ௦ㅰ࡟ࡼࡾ MCF ࢆ⏕ᡂࡍࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿ
㸦Fig.
3㸧ࠋ཯ᛂᛶ௦ㅰ≀࡛࠶ࡿ 2-PP ࡣ㸪ຍỈศゎ࡟ࡼࡾ⏕ᡂࡍࡿ MCF ࢆ௓ࡋ࡚⏕ᡂࡍࡿ࡜⪃࠼ࡽࢀࡿࡇ࡜࠿ࡽ㸪FBM ࡢ㓟໬௦ㅰ࡟ࡼࡿ 2OH-FBM ཬࡧ pOH-FBM ࡢ⏕ᡂࡣ㸪཯ᛂᛶ௦ㅰ
≀ࡢ⏕ᡂࢆᅇ㑊ࡋ㸪ຍỈศゎ࡟ࡼࡿ MCF ࡢ⏕ᡂࡣ཯ᛂᛶ௦ㅰ≀ࡢ⏕ᡂ࡟ࡘ࡞ࡀࡿ௦ㅰ⤒㊰࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋࡋ࠿ࡋ࡞ࡀࡽ㸪ࡇࢀࡽࡢ௦ㅰ࡟㛵୚ࡍࡿ⸆≀௦ㅰ㓝⣲࡟ࡘ࠸࡚ࡣ
ࡇࢀࡲ࡛ࡢ࡜ࡇࢁ༑ศ࡞᳨ウࡀ࡞ࡉࢀ࡚࠸࡞࠸ࠋ

ࡑࡇ࡛ᮏ❶࡛ࡣ㸪FBM ࡢ௦ㅰ࡟㛵୚ࡍࡿ⸆≀௦ㅰ㓝⣲ࢆ᫂ࡽ࠿࡟ࡍࡿࡇ࡜ࢆ┠ⓗ࡜ࡋ࡚
in vitro ௦ㅰヨ㦂ࢆᐇ᪋ࡋࡓࠋ

6

Fig. 3. Postulated metabolic pathway of FBM(Dieckhaus et al., 2001, 2002;; Kapetanovic et al., 2002)7

➨ 2 ⠇ ᐇ㦂᪉ἲI-2-i㸧 ໬ྜ≀ཬࡧヨ⸆

FBM ࡣ Sigma-Aldrich ࡼࡾ㉎ධࡋࡓࠋ⫢࣑ࢡࣟࢯ࣮࣒㸦ࣄࢺ㸪ࣛࢵࢺ㸪࣐࢘ࢫ㸧ࡣ Xenotechࡼࡾ㉎ධࡋࡓࠋࣂ࢟ࣗࣟ࢘࢕ࣝࢫឤᰁ᪻⹸⣽⬊⏤᮶ྛ✀ࣄࢺ CYP ศᏊ✀Ⓨ⌧⣔࣑ࢡࣟࢯ࣮࣒㸦CYP1A1㸪CYP1A2㸪CYP2A6㸪CYP2B6㸪CYP2C8㸪CYP2C9㸪CYP2C19㸪CYP2D6㸪CYP2E1㸪
CYP2J2 㸪 CYP3A4 㸪 CYP3A5 㸧 ཬ ࡧ ࣂ ࢟ ࣗ ࣟ ࢘ ࢕ ࣝ ࢫ ឤ ᰁ ᪻ ⹸ ⣽ ⬊ ⏤ ᮶ ྛ ✀ ࣄ ࢺcarboxylesterase㸦CES㸧ศᏊ✀Ⓨ⌧⣔࣑ࢡࣟࢯ࣮࣒㸦CES1b㸪CES1c㸪CES2㸧ࡣ CORNING
ࡼࡾ㉎ධࡋࡓࠋNADPH-regenerating system reagents ࡣ BD Biosciences ࡼࡾ㉎ධࡋࡓࠋࡑࡢ௚
ࡢヨ⸆ཬࡧ⁐፹ࡣ㧗㏿ᾮయࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮㸦high-performance liquid chromatography㸪
HPLC㸧⏝࠶ࡿ࠸ࡣ≉⣭ࡢᕷ㈍ရࢆ⏝࠸ࡓࠋ

I-2-ii㸧 ⫢࣑ࢡࣟࢯ࣮࣒ࢆ⏝࠸ࡓ FBM ࡢ௦ㅰヨ㦂ࣄࢺ㸪ࣛࢵࢺཬࡧ࣐࢘ࢫ⫢࣑ࢡࣟࢯ࣮࣒㸦᭱⤊⃰ᗘ࡜ࡋ࡚ 1 mg/mL㸧ࢆ㓝⣲※㸪NADPHࢆ⿵㓝⣲࡜ࡋ࡚㸪PBS㸦pH 7.4㸧୰࡛ FBM㸦᭱⤊⃰ᗘ࡜ࡋ࡚ 10 ࠶ࡿ࠸ࡣ 1ȝmol/L㸧ࢆ 37°C
᮲௳ୗ࡟࡚ 0㸪10㸪30㸪60㸪90 ཬࡧ 120 ศ㛫཯ᛂࡉࡏࡓࠋ཯ᛂᾮ࡜➼㔞ࡢị෭ࡋࡓ࢔ࢭࢺ
ࢽࢺࣜࣝࢆῧຍࡍࡿࡇ࡜࡛཯ᛂࢆ೵Ṇࡉࡏ㸪4°C㸪3000 rpm ࡛ 10 ศ㛫㐲ᚰศ㞳ࡋࡓࠋୖΎ
ࢆ⭷ࣇ࢕ࣝࢱ࣮ࢆ⏝࠸࡚㐲ᚰℐ㐣ᚋ㸪ℐᾮࢆ HPLC㸦Agilent 1290 Infinity㸪
Agilent Technologies㸧࡟ Orbitrap ㉁㔞ศᯒ⿦⨨㸦Orbitrap Velos Pro㸪Thermo Fisher Scientific㸧ࢆ᥋⥆ࡋࡓᾮయࢡࣟ
࣐ࢺࢢࣛࣇ࢕࣮ࢱࣥࢹ࣒㉁㔞ศᯒ⿦⨨㸦liquid chromatography-tandem mass spectrometry㸪LCMS㸧࡟ὀධࡋ㸪positive ion mode ࡟࡚ FBM㸪㓟໬௦ㅰ≀࡛࠶ࡿ OH-FBM ཬࡧຍỈศゎ௦ㅰ≀࡛࠶ࡿ MCF ࢆᐃࡋࡓࠋศᯒ࣒࢝ࣛࡣ ACQUITY UPLC BEH 18㸦ȝm, 2.1 × 100 mm㸪

Waters㸧ࢆ⏝࠸ࡓࠋ⛣ື┦࡟ࡣ㸪࣑ࣜ Q Ỉ㸸ࢠ㓟㸦1000㸸1㸪v/v㸧
㸦⛣ື┦ A㸧࡜࢔ࢭࢺࢽ
ࢺࣜࣝ㸸ࢠ㓟㸦1000㸸1㸪v/v㸧㸦⛣ື┦ B㸧ࢆ⏝࠸ࡓࠋ⛣ື┦ࡢ໙㓄ࡣ㸪ὀධᚋ 0 min ࠿ࡽ
3 min ࡲ࡛⛣ື┦ B ࡢ๭ྜࢆ 5%࡟ಖࡕ㸪3 min ࠿ࡽ 12 min ࡲ࡛ 5%࠿ࡽ 70%ࡲ࡛┤⥺ⓗ࡟8ୖ᪼ࡉࡏ㸪12 min ࠿ࡽ 15 min ࡲ࡛ 70%࡜୍ᐃ࡜ࡋࡓࠋ

I-2-iii㸧 CYP ཬࡧ CES Ⓨ⌧⣔࣑ࢡࣟࢯ࣮࣒ࢆ⏝࠸ࡓ⸆≀௦ㅰ㓝⣲ࡢ᳨ウࣄࢺ CYP Ⓨ⌧⣔࣑ࢡࣟࢯ࣮࣒㸦᭱⤊⃰ᗘ࡜ࡋ࡚ 100 pmol/mL㸧࠶ࡿ࠸ࡣࣄࢺ CES Ⓨ⌧⣔࣑ࢡࣟࢯ࣮࣒㸦᭱⤊⃰ᗘ࡜ࡋ࡚ 1 mg/mL㸧ࢆ㓝⣲※㸪NADPH ࢆ⿵㓝⣲࡜ࡋ࡚㸪PBS㸦pH
7.4㸧୰࡛ FBM㸦᭱⤊⃰ᗘ࡜ࡋ࡚ 1 ȝmol/L㸧ࢆ 37°C㸪120 ศ㛫཯ᛂࡉࡏࡓࠋ120 ศᚋ࡟཯ᛂᾮ࡜➼㔞ࡢị෭ࡋࡓ࢔ࢭࢺࢽࢺࣜࣝࢆῧຍࡍࡿࡇ࡜࡛཯ᛂࢆ೵Ṇࡉࡏ㸪4°C㸪3000 rpm ࡛
10 ศ㛫㐲ᚰศ㞳ࡋࡓࠋୖΎࢆ⭷ࣇ࢕ࣝࢱ࣮ࢆ⏝࠸࡚㐲ᚰℐ㐣ᚋ㸪ℐᾮࢆ HPLC㸦Agilent 1290
Infinity㸪Agilent Technologies㸧࡟ Orbitrap ㉁㔞ศᯒ⿦⨨㸦Orbitrap Velos Pro㸪Thermo Fisher
Scientific㸧ࢆ᥋⥆ࡋࡓ LC-MS ࡟ὀධࡋ㸪positive ion mode ࡟࡚ṧᏑࡍࡿ FBM 㔞ࢆᐃࡋࡓࠋศᯒ࣒࢝ࣛࡣ ACQUITY UPLC BEH 18㸦ȝm, 2.1 × 100 mm㸪Waters㸧ࢆ⏝࠸ࡓࠋ⛣ື┦࡟ࡣ㸪࣑ࣜ Q Ỉ㸸ࢠ㓟㸦1000㸸1㸪v/v㸧
㸦⛣ື┦ A㸧࡜࢔ࢭࢺࢽࢺࣜࣝ㸸ࢠ㓟㸦1000㸸1㸪
v/v㸧
㸦⛣ື┦ B㸧ࢆ⏝࠸ࡓࠋ⛣ື┦ࡢ໙㓄ࡣ㸪ὀධᚋ 0 min ࠿ࡽ 3 min ࡲ࡛⛣ື┦ B ࡢ๭ྜࢆ 5%࡟ಖࡕ㸪3 min ࠿ࡽ 12 min ࡲ࡛ 5%࠿ࡽ 70%ࡲ࡛┤⥺ⓗ࡟ୖ᪼ࡉࡏ㸪
12 min ࠿ࡽ 15 minࡲ࡛ 70%࡜୍ᐃ࡜ࡋࡓࠋ

I-2-iv㸧 ⤫ィゎᯒCYP ཬࡧ CES Ⓨ⌧⣔࣑ࢡࣟࢯ࣮࣒ࢆ⏝࠸ࡓ⸆≀௦ㅰ㓝⣲ࡢ᳨ウ㸦I-2-iii㸧࡟࠾࠸࡚㸪᭷ពᕪࡢ᳨ᐃࡣ Dunnett ᳨ᐃ࡟ࡼࡾᐇ᪋ࡋࡓࠋp ್ࡀ 0.05 ᮍ‶ࢆ⤫ィⓗ࡟᭷ព࡜ࡳ࡞ࡋࡓࠋ

9➨ 3 ⠇ ⤖ᯝ

I-3-i㸧 ⫢࣑ࢡࣟࢯ࣮࣒ࢆ⏝࠸ࡓ FBM ࡢ௦ㅰヨ㦂FBM㸦1ȝmol/L㸧ࢆ NADPH ࡢᏑᅾୗ࡛ࣄࢺ⫢࣑ࢡࣟࢯ࣮࣒࡜ 0㸪10㸪30㸪60㸪90 ཬࡧ
120 ศ㛫࢖࣮ࣥ࢟ࣗ࣋ࢩࣙࣥࡋࡓ࡜ࡇࢁ㸪࣑ࢡࣟࢯ࣮࣒୰ࡢ FBM ࡢ㔞ࡣ࢖࣮ࣥ࢟ࣗ࣋ࢩࣙ
ࣥ᫬㛫ࡢቑຍ࡟కࡗ࡚పୗࡍࡿഴྥࡀ☜ㄆࡉࢀࡓ㸦Fig. 4㸧ࠋࡇࡢ⤖ᯝࢆ㋃ࡲ࠼㸪௨㝆ࡢᐇ㦂ࡣ༑ศ࡟཯ᛂࡀ㐍ࢇ࡛࠸ࡿ࡜⪃࠼ࡽࢀࡿ࢖࣮ࣥ࢟ࣗ࣋ࢩࣙࣥ᫬㛫 120 ศ࡛ᐇ᪋ࡋࡓࠋ

FBM(nmol/mg protein)

12
10
8
6
4
2
0
0

20 40 60 80 100 120
Time (min)Fig. 4. Time course of in vitro metabolism of FBM in human liver microsmes.
FBM were incubated with human liver microsomes in the presence of NADPH
for 0, 10, 30, 60, 90 and 120 min. Amount of FBM were monitored by LC-MS.
Data represent the mean ± SD (n=3).

FBM ࡢ㓟໬ཬࡧຍỈศゎࡢ✀ᕪ࡟ࡘ࠸᳨࡚ウࡍࡿࡓࡵ㸪ࣄࢺ㸪ࣛࢵࢺཬࡧ࣐࢘ࢫ⫢࣑ࢡࣟࢯ࣮࣒ࢆ⏝࠸᳨࡚ウࢆ⾜ࡗࡓࠋFBM㸦1ȝmol/L㸧ࢆ NADPH ࡢᏑᅾୗ࡛ࣄࢺ㸪ࣛࢵࢺཬࡧ࣐࢘ࢫ⫢࣑ࢡࣟࢯ࣮࣒࡜ 120
ศ㛫࢖࣮ࣥ࢟ࣗ࣋ࢩࣙࣥࡋࡓ࡜ࡇࢁ㸪඲࡚ࡢ⫢࣑ࢡࣟࢯ࣮࣒୰࠿ࡽ㓟໬௦ㅰ≀࡛࠶ࡿ OHFBM ࡀ᳨ฟࡉࢀࡓ㸦Fig. 5㸧ࠋࡲࡓ㸪ຍỈศゎ௦ㅰ≀࡛࠶ࡿ MCF ࡣࣄࢺ࡜࣐࢘ࢫࡢ⫢࣑ࢡࣟࢯ࣮࣒୰࠿ࡽࡣ᳨ฟࡉࢀࡓ୍᪉㸪ࣛࢵࢺ⫢࣑ࢡࣟࢯ࣮࣒୰࠿ࡽࡣ᳨ฟࡉࢀ࡞࠿ࡗࡓ㸦Fig.
5㸧
ࠋ10

OH-FBM

MCF

N.D.

Fig. 5. In vitro metabolism of FBM in human, rat and mouse liver microsomes with NADPH.
FBM were incubated with human (upper column), rat (middle column) and mouse (lower column)
liver microsomes in the presence of NADPH for 120 min. Formation of OH-FBM and MCF weremonitored by LC-MS. N.D.: Not detected11ḟ࡟ NADPH ࡢ㠀Ꮡᅾୗ࡛ࣄࢺ㸪ࣛࢵࢺཬࡧ࣐࢘ࢫ⫢࣑ࢡࣟࢯ࣮࣒ࢆ⏝࠸࡚ྠᵝࡢ᳨ウࢆ⾜ࡗࡓࡇࢁ㸪OH-FBM ࡣࣄࢺ㸪ࣛࢵࢺ㸪࣐࢘ࢫ࠸ࡎࢀࡢ⫢࣑ࢡࣟࢯ࣮࣒୰࠿ࡽࡶ᳨ฟࡉ
ࢀ࡞࠿ࡗࡓ㸦Fig. 6㸧ࠋ୍᪉࡛㸪MCF ࡣ᳨ウࢆ⾜ࡗࡓ඲࡚ࡢ⫢࣑ࢡࣟࢯ࣮࣒୰࠿ࡽ᳨ฟࡉࢀ
ࡓ㸦Fig. 6㸧
ࠋ

N.D.

N.D.

N.D.

MCF

Fig. 6. In vitro metabolism of FBM in human, rat and mouse liver microsomes without NADPH.
FBM were incubated with human (upper column), rat (middle column) and mouse (lower column)
liver microsomes in the absence of NADPH for 120 min. Formation of OH-FBM and MCF weremonitored by LC-M
MS. N.D.: Not detected

12

FBM ࢆ NADPH ࡢᏑᅾୗཬࡧ㠀Ꮡᅾୗ࡛ࣄࢺ㸪ࣛࢵࢺ㸪࣐࢘ࢫ⫢࣑ࢡࣟࢯ࣮࣒࡜࢖ࣥ࢟
࣮ࣗ࣋ࢩࣙࣥࡋࡓ㝿ࡢ OH-FBM ཬࡧ MCF ࡢ LC-MS ศᯒ࡟࠾ࡅࡿࣆ࣮ࢡ㠃✚್ࢆ Table 1࡟♧ࡍࠋTable 1.. Comparison of MS peak area of OH--FBM and MCF in human, rat and mouse liver
microsomes with or without NADPH.
(A)
Biologicalmaterials

Livermicrosomes

Species

Cofactor

MS Peak Area of
OH FBM

Peak Area Ratio of
OH-FBM(Animal/Human)

MS Peak Area of
MCF

Peak Area Ratio of
MCF(Animal/Human)

Human

NADPH

119691

1

4843

1

Rat

NADPH

329660

2.75

N.D.

-

Mouse

NADPH

338606

2.83

5909

1.22

Species

Cofactor

MS Peak Area of
OH FBM

Peak Area Ratio of
OH-FBM(Animal/Human)

MS Peak Area of
MCF

Peak Area Ratio of
MCF(Animal/Human)

Human

-

N.D.

-

6937

1

Rat

-

N.D.

-

3895

0.56

Mouse

-

N.D.

-

7316

1.05

(B)
Biologicalmaterials

Livermicrosomes

FBM were incubated with human, rat and mouse liver microsomes in the presence (A) or
absence (B) of NADPH for 120 min. Formation of OH-FBM and MCF were monitored by LCMS. N.D.: Not detectedࣄࢺ㸪ࣛࢵࢺཬࡧ࣐࢘ࢫ⫢࣑ࢡࣟࢯ࣮࣒ࢆ⏝࠸ࡓ᳨ウ࡟࠾࠸࡚㸪㓟໬௦ㅰ≀࡛࠶ࡿ OHFBM ࡣ NADPH ࡢᏑᅾୗ᳨࡛ฟࡉࢀ㸪ࡑࡢ⏕ᡂ㔞ࡣࣄࢺ࡜ẚ㍑ࡋ࡚ࣛࢵࢺཬࡧ࣐࢘ࢫ࡛⣙3 ಸ⛬ᗘ㧗࠸ࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓ㸦Table 1㸦A㸧㸧ࠋOH-FBM ࡣ NADPH ࡢ㠀Ꮡᅾୗ࡛ࡣ᳨
ฟࡉࢀࡎ㸪ࡲࡓ㸪ຍỈศゎ௦ㅰ≀࡛࠶ࡿ MCF ࡣ NADPH ࡢ㠀Ꮡᅾୗ࡟࠾࠸࡚ࡶ඲࡚ࡢ⫢࣑ࢡࣟࢯ࣮࣒୰࠿ࡽ᳨ฟࡉࢀࡓ㸦Table 1㸦B㸧㸧ࠋࡉࡽ࡟㸪NADPH ࡢᏑᅾୗཬࡧ㠀Ꮡᅾୗ࡟࠾
࠸࡚⏕ᡂࡋࡓ MCF ࡢࣆ࣮ࢡ㠃✚್ࢆẚ㍑ࡍࡿ࡜㸪࠸ࡎࢀࡢ⫢࣑ࢡࣟࢯ࣮࣒࡟࠾࠸࡚ࡶ㸪
NADPH Ꮡᅾୗ࡜ẚ㍑ࡋ࡚ NADPH ࡢ㠀Ꮡᅾୗ࡟࠾࠸࡚ MCF ࡢ⏕ᡂ㔞ࡀቑຍഴྥࢆ♧ࡍࡇ࡜ࡀ☜ㄆࡉࢀࡓ㸦Table 1㸦A㸧
㸪㸦B㸧
㸧ࠋ

13

ࡇࢀࡽࡢ⤖ᯝࡼࡾ㸪FBM ࡢ㓟໬௦ㅰ࡟ࡣ NADPH ࢆᚲせ࡜ࡍࡿ㓟໬௦ㅰ㓝⣲࡜ࡋ࡚ᗈࡃ
▱ࡽࢀ࡚࠸ࡿ CYP ࡀ㛵୚ࡋ㸪ຍỈศゎ௦ㅰ࡟ࡣ NADPH ࢆᚲせ࡜ࡋ࡞࠸࣑ࢡࣟࢺ࣮࣒⏬ศ࡟ᒁᅾࡍࡿຍỈศゎ㓝⣲࡜ࡋ࡚ᗈࡃ▱ࡽࢀ࡚࠸ࡿ CES ࡀ㛵୚ࡍࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋࡲ
ࡓ㸪㓟໬௦ㅰࡀ㉳ࡇࡽ࡞࠸࡜⪃࠼ࡽࢀࡿ NADPH 㠀Ꮡᅾୗࡢ᮲௳࡟࠾࠸࡚ MCF ࡢ⏕ᡂ㔞ࡀቑຍഴྥࢆ♧ࡋࡓࡇ࡜࠿ࡽ㸪㓟໬௦ㅰࡀ㜼ᐖࡉࢀࡿࡇ࡜࡛ 2-PP ࡢ⏕ᡂ࡟ࡘ࡞ࡀࡿ MCF ࡢ⏕ᡂࡀቑຍࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ

14

I-3-ii㸧 CYP ཬࡧ CES Ⓨ⌧⣔࣑ࢡࣟࢯ࣮࣒ࢆ⏝࠸ࡓ⸆≀௦ㅰ㓝⣲ࡢ᳨ウ

FBM ࡢ㓟໬ཬࡧຍỈศゎ࡟㛵୚ࡍࡿ⸆≀௦ㅰ㓝⣲࡟ࡘ࠸࡚ࡉࡽ࡟᳨ウࡍࡿࡓࡵ㸪ࣄࢺ
CYP Ⓨ⌧⣔࣑ࢡࣟࢯ࣮࣒ཬࡧࣄࢺ CES Ⓨ⌧⣔࣑ࢡࣟࢯ࣮࣒ࢆ⏝࠸᳨࡚ウࢆ⾜ࡗࡓࠋFBM㸦10 ȝmol/L㸧ࢆࣄࢺࡢྛ CYP ศᏊ✀ࢆⓎ⌧ࡉࡏࡓ࣑ࢡࣟࢯ࣮࣒࡜࢖࣮ࣥ࢟ࣗ࣋ࢩ
ࣙࣥࡋࡓ࡜ࡇࢁ㸪120 ศᚋࡢ FBM ࡢṧᏑ㔞ࡣ CYP2C8㸪CYP2C9 ཬࡧ CYP2E1 Ⓨ⌧࣑ࢡࣟࢯ࣮࣒࡛᭷ព࡟పୗࡋࡓ㸦Fig. 7㸧
ࠋ

Fig. 7. In vitro metabolism of FBM in recombinant human CYP expressing microsomes.
FBM were incubated with human CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9,,CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4 and CYP3A5 expressing microsomes for
120 min. Remaining amount of FBM were measured by LC-MS. Data represent the mean ±SD (n=3). *p<0.05 vs control microsomes.

ࡲࡓ㸪FBM ࢆࣄࢺࡢྛ CES ศᏊ✀ࢆⓎ⌧ࡉࡏࡓ࣑ࢡࣟࢯ࣮࣒࡜࢖࣮ࣥ࢟ࣗ࣋ࢩࣙࣥࡋ
ࡓ࡜ࡇࢁ㸪120 ศᚋࡢ FBM ࡢṧᏑ㔞ࡣ CES1c Ⓨ⌧࣑ࢡࣟࢯ࣮࣒࡟࠾࠸࡚᭷ព࡟పୗࡋࡓ
㸦Fig. 8㸧ࠋ

15

Fig. 8. In vitro metabolism of FBM in recombinant human CES expressing microsomes.
FBM were incubated with human CES1b, CES1c and CES2 expressing microsomes for 120 min.
Remaining amount of FBM were measured by LC-MS. Data represent the mean ± SD (n=3).
*p<0.05 vs control microsomes.

௨ୖࡢ⤖ᯝ࠿ࡽ㸪FBM ࡢ㓟໬௦ㅰ࡟ࡣ CYP2C8㸪CYP2C9 ཬࡧ CYP2E1 ࡀ㛵୚ࡋ㸪ຍỈศゎ௦ㅰ࡟ࡣ CES1c ࡀ㛵୚ࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ

16

➨ 4 ⠇ ᑠᣓᮏ❶࡛ࡣ㸪⫢࣑ࢡࣟࢯ࣮࣒㸪ࣄࢺ CYP Ⓨ⌧⣔࣑ࢡࣟࢯ࣮࣒ཬࡧ CES Ⓨ⌧⣔࣑ࢡࣟࢯ࣮
࣒ࢆ⏝࠸࡚ FBM ࡢ௦ㅰ࡟㛵୚ࡍࡿ⸆≀௦ㅰ㓝⣲ࡢ᳨ウࢆ⾜ࡗࡓࠋ⫢࣑ࢡࣟࢯ࣮࣒ࢆ⏝࠸ࡓ᳨ウ࡟࠾࠸࡚㸪㓟໬௦ㅰ≀࡛࠶ࡿ OH-FBM ࡣ NADPH ࡢᏑᅾୗ࡛⏕ᡂࡍࡿ୍᪉㸪NADPH ࡢ㠀Ꮡᅾୗ࡛ࡣ⏕ᡂࡋ࡞࠸ࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓ㸦Fig. 5㸪Fig.
6㸧ࠋࡲࡓ㸪ຍỈศゎ௦ㅰ≀࡛࠶ࡿ MCF ࡣ㸪NADPH ࡢ㠀Ꮡᅾୗ࡟࠾࠸࡚ࡶ⏕ᡂࡍࡿࡇ࡜ࡀ
☜ㄆࡉࢀࡓ㸦Fig. 6㸧ࠋࡇࢀࡽࡢ⤖ᯝࡼࡾ㸪FBM ࡢ㓟໬௦ㅰ࡟ࡣ NADPH ࢆᚲせ࡜ࡍࡿ௦ㅰ
㓝⣲࡛࠶ࡿ CYP㸪ຍỈศゎ௦ㅰ࡟ࡣ NADPH ࢆᚲせ࡜ࡋ࡞࠸௦ㅰ㓝⣲࡛࠶ࡿ CES ࡢ㛵୚ࡀ♧၀ࡉࢀࡓࠋ
ࡲࡓ㸪MCF ࡢ⏕ᡂ㔞ࡣ㸪NADPH Ꮡᅾୗ࡜ẚ㍑ࡋ࡚㸪㓟໬௦ㅰࡀ㉳ࡇࡽ࡞࠸࡜⪃࠼ࡽࢀ
ࡿ NADPH 㠀Ꮡᅾୗ࡟࠾࠸࡚ቑຍഴྥࢆ♧ࡋࡓ㸦Table 1㸧ࠋᮏ◊✲࡟࠾࠸࡚ࡣ㸪⫢࣑ࢡࣟࢯ࣮࣒୰࡛཯ᛂᛶ௦ㅰ≀࡛࠶ࡿ 2-PP ࡣ᳨ฟࡉࢀ࡚࠸࡞࠸㸦data not shown㸧ࠋ2-PP ࡀ Fig.4 ࡟♧ ࡍ ከ ẁ 㝵 ࡢ ཯ ᛂ ࢆ ⤒ ࡚ ⏕ ᡂ ࡍ ࡿ ࡇ ࡜ ࡸ 㸪 2-PP ࡢ ཯ ᛂ ᛶ ࡀ 㧗 ࠸ ࡇ ࡜ ࢆ ㋃ ࡲ ࠼ ࡿ ࡜㸦Dieckhaus et al.㸪2001㸹Thompson et al.㸪1996㸧
㸪in vitro ᮲௳ୗ࡛ 2-PP ࢆ᳨ฟࡋ㸪ࡑࡢኚືࢆゎᯒࡍࡿࡇ࡜ࡣᅔ㞴࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋࡋ࠿ࡋ࡞ࡀࡽ㸪NADPH 㠀Ꮡᅾୗ࡛ MCF ࡢ⏕ᡂ㔞ࡀቑຍࡋࡓ⤖ᯝࡣ㸪FBM ࢆᢞ୚ࡋࡓ㝿࡟㸪ఱࡽ࠿ࡢせᅉ࡛㓟໬௦ㅰࡀ㜼ᐖࡉࢀࡿ࡜㸪
MCF ࡢ⏕ᡂ㔞ࡀቑຍࡋ㸪2-PP ࡢቑຍ࡟ࡘ࡞ࡀࡿྍ⬟ᛶࢆ♧၀ࡋ࡚࠸ࡿࡶࡢ࡜⪃࠼ࡽ࠼ࢀࡓࠋࡉࡽ࡟㸪MCF ࡣࣄࢺ࡟࠾࠸࡚ NADPH ࡢᏑᅾୗ᳨࡛ฟࡉࢀ࡚࠸ࡿ୍᪉㸪ࣛࢵࢺ࡟࠾࠸࡚
ࡣ NADPH ࡢᏑᅾୗ࡛ࡣ᳨ฟࡉࢀ࡚࠸࡞࠸㸦Fig. 5㸧ࠋࡇࡢ⤖ᯝࡣ㸪ࣛࢵࢺ࡟࠾࠸࡚ࡣ㏻ᖖ
ࡢ௦ㅰ཯ᛂ᮲௳ୗ࡛ࡣ㸪ຍỈศゎ௦ㅰ࡟ẚ࡭࡚㓟໬௦ㅰࡀඃ఩࡟ാ࠸࡚࠸ࡿࡇ࡜ࢆ♧၀ࡋ࡚࠸ࡿ࡜⪃࠼ࡽࢀ㸪ࡇࡢࡇ࡜ࡀ㸪FBM ࡢẘᛶヨ㦂࡟࠾࠸࡚⫢ẘᛶࡀ᳨ฟࡉࢀ࡞࠿ࡗࡓࡇ࡜
࡟୍㒊㛵୚ࡋ࡚࠸ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋ

17ࣄࢺ CYP ཬࡧ CES Ⓨ⌧⣔࣑ࢡࣟࢯ࣮࣒ࢆ⏝࠸ࡓ᳨ウ࡟࠾࠸࡚㸪࢖࣮ࣥ࢟ࣗ࣋ࢩࣙࣥ 120
ศᚋࡢ FBM ࡢṧᏑ㔞ࡣ CYP2C8㸪CYP2C9㸪CYP2E1 Ⓨ⌧࣑ࢡࣟࢯ࣮࣒ཬࡧ CES1c Ⓨ⌧࣑ࢡࣟࢯ࣮࣒࡛᭷ព࡟పୗࡋࡓ㸦Fig. 7㸪Fig. 8㸧ࠋࡇࢀࡽࡢ⤖ᯝࡼࡾ㸪
FBM ࡢ㓟໬௦ㅰ࡟ CYP2C8㸪
CYP2C9 ཬࡧ CYP2E1 ࡀ㛵୚ࡋ㸪ຍỈศゎ௦ㅰ࡟ CES1c ࡀ㛵୚ࡍࡿࡶࡢ࡜⪃࠼ࡽࢀࡓࠋࡲ
ࡓ㸪CES1c ࢆ௓ࡋࡓຍỈศゎ௦ㅰࡀ 2-PP ࡢ⏕ᡂ࡟㛵୚ࡋ㸪CYP2C8㸪CYP2C9 ཬࡧ CYP2E1
ࢆ௓ࡋࡓ㓟໬௦ㅰࡀ 2-PP ࡢ⏕ᡂࢆᅇ㑊ࡍࡿ㔜せ࡞௦ㅰ⤒㊰࡜࡞ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ
୍⯡ⓗ࡟㸪⸆≀ࡢ௦ㅰ࡟㛵୚ࡍࡿ㓝⣲ࢆ᳨ウࡍࡿ㝿ࡣ㸪㓝⣲ࡢ㜼ᐖ๣ࡸศᏊ✀Ⓨ⌧⣔ࢆ⏝࠸ࡓ᳨ウࡀ⾜ࢃࢀࡿࠋᮏ◊✲࡛ࡣ㸪ศᏊ✀Ⓨ⌧⣔࡛࠶ࡿࣄࢺ CYP ཬࡧ CES Ⓨ⌧⣔࣑ࢡࣟࢯ࣮࣒ࢆ⏝࠸᳨࡚ウࢆ⾜ࡗࡓࠋGlue ࡽ㸦1997㸧ࡣ CYP ࡢ㜼ᐖ๣ࢆ⏝࠸ࡓ᳨ウ࠿ࡽ㸪FBM
ࡢ௦ㅰ࡟ CYP3A4 ཬࡧ 2E1 ࡀ㛵୚ࡍࡿࡇ࡜ࢆሗ࿌ࡋ࡚࠸ࡿࠋᮏ◊✲࡟࠾࠸࡚㸪FBM ࡢṧᏑ㔞ࡣ CYP2E1 Ⓨ⌧࣑ࢡࣟࢯ࣮࣒࡛᭷ព࡟పୗࡋࡓࡶࡢࡢ㸪CYP3A4 Ⓨ⌧࣑ࢡࣟࢯ࣮࣒࡛ࡣ
పୗࡋ࡞࠿ࡗࡓࠋࡇࡢ஋㞳࡟ࡘ࠸࡚ࡣ⌧ᅾࡢ࡜ࡇࢁ୙࡛᫂࠶ࡿࡶࡢࡢ㸪CYP Ⓨ⌧⣔࡜ CYP㜼ᐖ๣࡜࠸࠺཮᪉ࡢヨ㦂᪉ἲࡢ㐪࠸ࡀཎᅉ࡛࠶ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋ

ࡇࢀࡲ࡛ࡢ◊✲࠿ࡽ㸪CYP2C ศᏊ✀ࡢⓎ⌧࡟ࡣ✀ᕪࡀ࠶ࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠾ࡾ㸪ࣄࢺ࡟ẚ࡭࡚ࡆࡗṑ㢮࡛ࡣ Cyp2c ࡢⓎ⌧㔞ࡀ㧗ࡃ㸪ࡆࡗṑ㢮࡟࠾࠸࡚ࡣ Cyp2c ࡀ୺せ࡞⸆≀௦ㅰ㓝⣲࡛࠶ࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ㸦Martignoni et al.㸪2006㸧
ࠋࡇࡢࡼ࠺࡞ CYP2C ศᏊ✀ࡢⓎ⌧㔞ࡢ㐪࠸ࡀ㸪⫢࣑ࢡࣟࢯ࣮࣒ࢆ⏝࠸ࡓ᳨ウ࡟࠾࠸࡚㸪ࣄࢺ࡜ẚ࡭࡚ࣛࢵࢺཬࡧ࣐࢘ࢫ
࡛ OH-FBM ࡢ⏕ᡂ㔞ࡀከ࠿ࡗࡓࡇ࡜࡟㛵ಀࡋ࡚࠸ࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿ㸦Table 1㸧ࠋ
CES1 ࡣࣄࢺࡢ⫢⮚࡟᭱ࡶ㇏ᐩ࡟Ꮡᅾࡍࡿ⸆≀௦ㅰ㓝⣲ࡢ 1 ࡘ࡛࠶ࡿ୍᪉㸪CES2 ࡣ୺࡟ᑠ⭠࡟Ⓨ⌧ࡋ࡚࠸ࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ㸦Di㸪2019㸧ࠋᚑࡗ࡚㸪ࣄࢺ࡟࠾࠸࡚ẘᛶⓎ⌧⮚ჾ࡜ྠࡌ⫢⮚࡟Ⓨ⌧ࡍࡿ CES1c ࢆ௓ࡋ࡚ MCF ࢆ⏕ᡂࡍࡿࡶࡢ࡜⪃࠼ࡽࢀࡓࠋ
ᮏ◊✲࡛ࡣ FBM ࡢຍỈศゎ࡟㛵୚ࡍࡿࡆࡗṑ㢮ࡢ CES ศᏊ✀࡟ࡘ࠸࡚ࡣ᳨ウࡋ࡚࠸࡞࠸ࠋࡆࡗṑ㢮࡛ࡣࣄࢺ࡜ẚ࡭࡚ከࡃࡢ CES ศᏊ✀ࡀⓎ⌧ࡋ࡚࠸ࡿࡇ࡜ࡸ㸦Di㸪2019㸧㸪ࣄࢺ࡜ࡆࡗṑ㢮ࡢ CES ࡢ┦ྠᛶࡀ 67-77%࡛࠶ࡿ࡜ሗ࿌ࡉࢀ࡚࠸ࡿࡇ࡜ࢆ㋃ࡲ࠼ࡿ࡜㸦Taketani
et al.㸪2007㸹Hosokawa㸪2008㸧㸪FBM ࡢຍỈศゎ࡟㛵୚ࡍࡿ CES ࡢศᏊ✀ࡣ㸪ࣄࢺ࡜ࡆࡗ

18ṑ㢮࡛␗࡞ࡿྍ⬟ᛶࡶ⪃࠼ࡽࢀࡿࠋ
CYP2C9 ࡟ࡣ㑇ఏᏊከᆺࡀᏑᅾࡍࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠾ࡾ㸪tolbutamide㸪phenytoin㸪warfarin࡞࡝㸪ᵝࠎ࡞⸆≀ࡢ௦ㅰࡀ㑇ఏᏊከᆺࡢᙳ㡪ࢆཷࡅࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ㸦Goldstein㸪
2001㸧ࠋࡲࡓ CES ࡢάᛶࡣಶయ㛫ᕪࡀ኱ࡁ࠸ࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ㸦Di㸪2019㸧ࠋࡇࡢࡼ࠺࡞᝟ሗࢆ㋃ࡲ࠼ࡿ࡜㸪FBM ࡢᢞ୚࡟ࡼࡿ 2-PP ࡢ⏕ᡂࡣ㸪CYP2C8㸪CYP2C9㸪
CYP2E1 ࢆ௓ࡋࡓ⸆≀㛫┦஫స⏝㸪CYP2C9 ࡢ㑇ఏᏊከᆺ㸪CES1c άᛶࡢಶయ㛫ᕪ࡞࡝࡟ࡼࡾᙳ㡪ࢆཷࡅࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋ
ᚑࡗ࡚㸪FBM ࡢ⮫ᗋ౑⏝࡟࠾࠸࡚㸪2-PP ࡢ⏕ᡂࢆᅇ㑊ࡍࡿ CYP2C8㸪CYP2C9 ཬࡧ CYP2E1
࡛௦ㅰࡉࢀࡿ⸆≀࡜ࡢే⏝㸪࠶ࡿ࠸ࡣ CYP2C9 ࡢ㑇ఏᏊከᆺࢆ᭷ࡍࡿᝈ⪅࡬ࡢᢞ୚ࡣ㸪
FBM
࡟ࡼࡿ⫢ẘᛶࡢࣜࢫࢡせᅉ࡟࡞ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ

19➨ II ❶ ࣄࢺ⫢⣽⬊࣓࣐࢟ࣛ࢘ࢫࢆ⏝࠸ࡓ FBM ࡢ௦ㅰ≀ࣉࣟࣇ࢓࢖ࣝཬࡧẘᛶホ౯➨ 1 ⠇ ⥴ゝ୍⯡ⓗ࡟㸪⸆≀ࡢࣄࢺ࡟࠾ࡅࡿ௦ㅰ≀ࣉࣟࣇ࢓࢖ࣝࢆ᳨ウࡍࡿࡓࡵ࡟㸪ࣄࢺ෾⤖⫢⣽⬊ࢆ⏝࠸ࡓ in vitro ௦ㅰヨ㦂ࡀ⾜ࢃࢀ࡚࠸ࡿࠋࡋ࠿ࡋ㸪ࡇࡢᡭἲ࡟ࡼࡿࣄࢺ௦ㅰ≀ࡢண⢭ᗘࡣ㸪㓟໬㸪㑏ඖ࡞࡝ࡢ➨ 1 ┦௦ㅰ཯ᛂ࡛ 50-69%㸪ࢢࣝࢡࣟࣥ㓟ᢪྜ㸪◲㓟ᢪྜ࡞࡝ࡢ➨ 2┦௦ㅰ཯ᛂ࡛ 47%࡛࠶ࡾ㸪≉࡟ከẁ㝵ࡢ཯ᛂࢆ⤒⏤ࡋ࡚⏕ᡂࡍࡿ௦ㅰ≀ࡢண⢭ᗘࡣప࠸ࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ㸦Dalvie et al.㸪2008㸧ࠋ
FBM ࡣ Fig. 3 ࡟♧ࡍ㏻ࡾ㸪2-PP ࢆྵࡵࡓከࡃࡢ௦ㅰ≀ࡀ➨ 1 ┦௦ㅰ཯ᛂ㸪➨ 2 ┦௦ㅰ཯ᛂࢆྵࡴከẁ㝵ࡢ௦ㅰ཯ᛂ࡟ࡼࡾ⏕ᡂࡍࡿ࡜⪃࠼ࡽࢀ࡚࠾ࡾ㸪ࣄࢺ෾⤖⫢⣽⬊ࢆ⏝࠸ࡓ invitro ௦ㅰヨ㦂࠿ࡽࡣ㸪FBM ࡢࣄࢺ࡛ࡢ௦ㅰ≀ࣉࣟࣇ࢓࢖ࣝࢆ༑ศ࡟᳨ドࡍࡿࡇ࡜ࡣ㞴ࡋ࠸࡜⪃࠼ࡽࢀࡿࠋ
ࣄࢺ࡟࠾ࡅࡿ௦ㅰ≀ࣉࣟࣇ࢓࢖ࣝࢆ᳨ウࡍࡿ᪉ἲ࡜ࡋ࡚㸪㏆ᖺ㸪ࣄࢺ໬ື≀ࣔࢹ࡛ࣝ࠶
ࡿࣄࢺ⫢⣽⬊࣓࣐࢟ࣛ࢘ࢫ㸦PXB ࣐࢘ࢫ㸹ᰴᘧ఍♫ࣇ࢙ࢽࢵࢡࢫࣂ࢖࢜㸧ࢆ⏝࠸ࡓ◊✲ࡀᩘከࡃሗ࿌ࡉࢀ࡚࠸ࡿ㸦De Serres et al.㸪2011㸹Inoue et al.㸪2009㸹Kamimura et al.㸪2010㸹
Yamazaki et al.㸪2010㸧ࠋ PXB ࣐࢘ࢫࡣ urokinase type plasminogen activator㸦uPA㸧㑇ఏᏊࢆ࣐࢘ࢫ࢔ࣝࣈ࣑࢚ࣥࣥ
ࣁࣥࢧ࣮࡟⤌ࡳ㎸ࡳ㸪⫢⮚≉␗ⓗ࡟Ⓨ⌧ࡉࡏࡿࡇ࡜࡛⫢㞀ᐖࢆⓎ⑕ࡋࡓ uPA ࣐࢘ࢫ࡜㸪ච␿୙඲ࡢᛶ㉁ࢆ᭷ࡍࡿ severe combined immnodefiviency disease㸦SCID㸧࣐࢘ࢫࢆ᥃ࡅྜࢃࡏ
ࡿࡇ࡜࡛㸪⫢㞀ᐖ࡜ච␿୙඲ࡢᛶ㉁ࢆేࡏᣢࡘ uPA/SCID ࣐࢘ࢫࢆసᡂࡋ㸪ࡇࡢ࣐࢘ࢫࢆ
࣍ࢫࢺ࡜ࡋ࡚ࣄࢺࡢ⫢⣽⬊ࢆ⛣᳜ࡍࡿࡇ࡜࡛సᡂࡉࢀࡿ㸦Tateno at al.㸪2015㸧
㸦Fig. 9㸧ࠋPXB
࣐࢘ࢫ࡟࠾࠸࡚ࡣ㸪࣐࢘ࢫ⫢⣽⬊ࡢ኱㒊ศࡀࣄࢺ⫢⣽⬊࡟⨨᥮ࡉࢀ࡚࠾ࡾ㸪ࡑࡢ⨨᥮⋡ࡣ
⣙ 80%࡜ሗ࿌ࡉࢀ࡚࠸ࡿ㸦Tateno at al.㸪2015㸧

20

HumanHepatocytes

uPA/SCID Mouse

Transplantation

Chimeric mice withhumanized liver

Liver injury
Immunodeficiency

Fig. 9. Production of chimeric mice with humanized liver (PXB-mmice).

 ࡇࢀࡲ࡛ࡢ◊✲࡟ࡼࡾ㸪PXB ࣐࢘ࢫ࡟࠾ࡅࡿ CYP ௦ㅰ㓝⣲ཬࡧ uridine 5’-diphosphoglucuronosyl transferase㸦UGT㸧ࡸ sulfotransferase㸦SULT㸧࡜࠸ࡗࡓ non-CYP ௦ㅰ㓝⣲ࡢⓎ⌧ࣞ࣋ࣝࡸάᛶࡣ㸪ࣄࢺ࡜ྠ⛬ᗘ࡛࠶ࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠾ࡾ㸦Katoh et al.㸪2004㸪2005㸹
Katoh and Yokoi㸪2007㸹Kitamura et al.㸪2008㸹Nishimura et al.㸪2005㸹Ohtsuki et al.㸪2014㸧㸪
PXB ࣐࢘ࢫࡣࣄࢺ࡟࠾ࡅࡿ⸆≀ືែࢆணࡍࡿࡓࡵࡢࣄࢺ໬ື≀ࣔࢹࣝ࡜ࡋ࡚ὀ┠ࡉࢀ࡚࠸ࡿࠋࡲࡓ㸪Kakuni ࡽ㸦2012㸧ࡣ㸪ࣄࢺ࡟࠾࠸࡚཯ᛂᛶ௦ㅰ≀࡟㉳ᅉࡍࡿ IDT ࡟ࡼࡾ⫢ẘᛶࢆ
Ⓨ⑕ࡍࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿ troglitazone ࢆ PXB ࣐࢘ࢫ࡟ᢞ୚ࡍࡿࡇ࡜࡛㸪ALT ཬࡧ AST ࡢ
ୖ᪼ࡀぢࡽࢀ㸪PXB ࣐࢘ࢫ࡟࠾࠸࡚⫢ẘᛶࡀⓎ⌧ࡋࡓࡇ࡜ࢆሗ࿌ࡋ࡚࠸ࡿࠋ
ࡇࢀࡽࡢሗ࿌ࢆ㋃ࡲ࠼ࡿ࡜㸪PXB ࣐࢘ࢫࡣࣄࢺ࡟࠾ࡅࡿ཯ᛂᛶ௦ㅰ≀ࡢ⏕ᡂࢆ௓ࡋࡓ⫢ẘᛶࢆホ౯ࡍࡿୖ࡛᭷⏝࡞ࣄࢺ໬ື≀ࣔࢹࣝ࡟࡞ࡿ࡜⪃࠼ࡽࢀࡿࠋ
FBM ࡣ㸪ࡑࡢ᥎ᐃ௦ㅰ⤒㊰㸦Fig. 3㸧ࡼࡾ㸪཯ᛂᛶ௦ㅰ≀࡛࠶ࡿ 2-PP ࡀ⏕ᡂࡋࡓࡢࡕ㸪
GSH ᢪྜࢆཷࡅࡿ࡜᥎ᐃࡉࢀࡿࠋࡋ࠿ࡋ࡞ࡀࡽ㸪཯ᛂᛶ௦ㅰ≀ࡢ⏕ᡂ࠿ࡽ⫢ẘᛶࡢⓎ⌧࡟⮳ࡿࡲ࡛ࡢ࣓࢝ࢽࢬ࣒ࢆ㸪⮫ᗋ࡛ࣄࢺࡢ⫢⮚ࢆ⏝࠸࡚┤᥋ⓗ࡟ホ౯ࡍࡿࡇ࡜ࡣᅔ㞴࡛࠶ࡿࡇ࡜࠿ࡽ㸪ࣄࢺ⫢⮚ࢆ᭷ࡍࡿ PXB ࣐࢘ࢫࡣ FBM ࡢẘᛶ࣓࢝ࢽࢬ࣒ࢆゎ᫂ࡍࡿୖ࡛᭷⏝࡞ື≀ࣔࢹࣝ࡜࡞ࡿࡇ࡜ࡀᮇᚅࡉࢀࡿࠋ

21⸆≀ࡀ⮚ჾ࡟୚࠼ࡿᙳ㡪ࢆ┤᥋ⓗ࡟ホ౯ࡍࡿ᪉ἲ࡜ࡋ࡚㸪㏆ᖺ㸪⤌⧊ษ∦ࢆ⏝࠸ࡓ㉁㔞
ศᯒ࢖࣓࣮ࢪࣥࢢἲ㸦mass spectrometry imaging㸪MSI㸧ࡀὀ┠ࡉࢀ࡚࠸ࡿ㸦Nishidate et al.㸪
2019㸧ࠋMSI ࡣ㸪ࣄࢺࡢ⏕య⤌⧊デ᩿࡟ࡼࡾᚓࡽࢀࡓ⮫ᗋࢧࣥࣉࣝ㸪࠶ࡿ࠸ࡣື≀ࡢ⮚ჾࡼࡾసᡂࡉࢀࡿ⤌⧊ษ∦ࢆ㉁㔞ศᯒ࡟౪ࡍࡿࡇ࡜࡛㸪ヨᩱ୰࡟ྵࡲࢀࡿ⸆≀ࡸ⏕యෆ≀㉁ࢆ
࢖࢜ࣥ໬ࡋ㸪ࡑࡢ఩⨨᝟ሗ࡜ేࡏ࡚ࢹ࣮ࢱྲྀᚓࡍࡿࡇ࡜࡟ࡼࡾ㸪⸆≀ࡸ⏕యෆ≀㉁ࡢศᕸ
ࢆ⏬ീ໬ࡍࡿᡭἲ࡛࠶ࡿ㸦Fig. 10㸧ࠋ
MSI ࡛ࡣ࢖࢜ࣥ໬ࡉࢀࡓศᏊࢆ㉁㔞ศᯒ⿦⨨᳨࡛ฟࡍࡿࡓࡵ㸪ᐃᑐ㇟≀㉁ࡢ m/z ࡀศ࠿ࢀࡤ㸪ᶆရࢆ⏝࠸ࡿࡇ࡜࡞ࡃ⤌⧊୰࡛ࡢศᕸࢆホ౯ࡍࡿࡇ࡜ࡀྍ⬟࡛࠶ࡿࠋࡇࢀࡲ࡛࡟ MSI ࢆ⏝࠸ࡓ᳨ウ࡟ࡼࡾ㸪⸆≀ཬࡧࡑࡢ௦ㅰ࡟ຍ࠼࡚㸪ෆᅉᛶ≀㉁࡛࠶ࡿࣜ
ࣥ⬡㉁ࡢศᕸࢆホ౯ྍ⬟࡛࠶ࡗࡓࡇ࡜㸦Sugiura et al.㸪2010㸹Takai et al㸪2015㸧ࠋࡀࢇᝈ⪅ࡢ⏕య⤌⧊デ᩿࡟ࡼࡾᚓࡽࢀࡓࢧࣥࣉࣝࢆ⏝࠸࡚㸪ෆᅉᛶ≀㉁࣍ࢫࣇ࢓ࢳࢪࣝ࢖ࣀࢩࢺ࣮
ࣝࡢศᕸࢆ㸪ࡀࢇ⤌⧊࡜ṇᖖ⤌⧊㒊ศ࡟ศࡅ࡚ゎᯒࡀྍ⬟࡛࠶ࡗࡓࡇ࡜㸦Goto et al.㸪2014㸹
Kawashima et al.㸪2013㸹Shimma et al.㸪2007㸧ࠋࡲࡓ㸪≉Ⓨᛶ⫵⥺⥔⑕἞⒪⸆ pirfenidone ࡢᢞ୚ᚋ࡟ኚືࡍࡿ⏕యෆࣂ࢖࣐࣮࣮࢜࢝ࡢ᳨ฟࡀྍ⬟࡛࠶ࡗࡓࡇ࡜࡞࡝ࡀሗ࿌ࡉࢀ࡚࠸ࡿ㸦Sun et al㸪2016㸧
ࠋ

MALDI-MSI(Visualization of drug andendogenous molecules)

Laser

Ქ

IonizationᲥᲥᲥ

Liver section on slide glass
Fig. 10.. Overv
view of MALDI--MSI.

22

ࡇ࠺ࡋࡓ⫼ᬒࢆ㋃ࡲ࠼㸪ᮏ❶࡛ࡣ㸪PXB ࣐࢘ࢫࢆ⏝࠸࡚ FBM ࢆᢞ୚ᚋࡢ௦ㅰ≀ࣉࣟࣇ࢓࢖ࣝࢆホ౯ࡍࡿ࡜࡜ࡶ࡟㸪MSI ࢆ⏝࠸࡚ FBM ࡢᢞ୚ࡀࣄࢺࡢ⫢⮚࡟୚࠼ࡿᙳ㡪࡟ࡘ࠸᳨࡚ウࢆ⾜ࡗࡓࠋ

23

➨ 2 ⠇ ᐇ㦂᪉ἲII-2-i㸧 ໬ྜ≀ཬࡧヨ⸆

FBM ࡣ Sigma-Aldrich ࠶ࡿ࠸ࡣ AK Scientifics ࡼࡾ㉎ධࡋࡓࠋ2,5-dihydroxybenzoic acid
㸦DHB㸧ࡣᮾி໬ᡂᕤᴗࡼࡾ㉎ධࡋࡓࠋࡑࡢ௚ࡢヨ⸆ཬࡧ⁐፹ࡣ HPLC ⏝࠶ࡿ࠸ࡣ≉⣭ࡢ
ᕷ㈍ရࢆ⏝࠸ࡓࠋ

II-2-ii㸧 ᐇ㦂ື≀

PXB ࣐࢘ࢫࡣ㸪Tateno ࡽ㸦2015㸧ࡢ᪉ἲ࡟ᚑࡗ࡚సᡂࡉࢀࡓࠋලయⓗ࡟ࡣ㸪cDNAuPAwild/+/SCID ࣐࢘ࢫࢆ࣍ࢫࢺ࡜ࡋ㸪ࣄࢺࡢ⫢⣽⬊ࢻࢼ࣮࡜ࡋ࡚ BD195㸦BD Biosciences㸧ࢆ⛣᳜ࡍࡿࡇ࡜࡟ࡼࡾసᡂࡋࡓࠋᐇ㦂࡟ࡣ⾑ᾮ୰ࡢࣄࢺ࢔ࣝࣈ࣑ࣥ⃰ᗘࡀ 7.0 mg/mL ௨ୖࡢ㞝ᛶࡢಶయࢆ⏝࠸ࡓࠋPXB ࣐࢘ࢫࡢసᡂཬࡧ໬ྜ≀ࡢᢞ୚㸪᥇ᮦࡣᰴᘧ఍♫ࣇ࢙ࢽࢵࢡࢫ
ࣂ࢖࢜࡟࡚ᢎㄆࡉࢀࡓᐇ㦂ィ⏬࡟ᚑࡗ࡚ᐇ᪋ࡉࢀࡓࠋ

II-2-iii㸧 PXB ࣐࢘ࢫ࡟࠾ࡅࡿ FBM ࡢ௦ㅰ≀ࣉࣟࣇ࢓࢖ࣝホ౯

FBM ࢆ 0.5%࢝ࣝ࣎࢟ࢩ࣓ࢳࣝࢭ࣮ࣝࣟࢫ࡟ᠱ⃮ࡋ࡚ 600 mg/10 mL/kg ࡛ PXB ࣐࢘ࢫ࡟⤒ཱྀᢞ୚ࡋ㸪2 ᫬㛫ᚋࡢ⾑₢ཬࡧ⫢⮚ࢆ᥇ྲྀࡋࡓ㸦n=3㸧ࠋ⫢⮚ࡣ PBS㸦pH 7.4㸧࡜ΰྜࡋ࡚
20%࣍ࣔࢪࢿ࣮ࢺࢆㄪ〇ࡋࡓࠋྛ⾑₢ཬࡧ 20%⫢࣍ࣔࢪࢿ࣮ࢺࢧࣥࣉࣝࢆ➼㔞ࡎࡘΰྜࡋ㸪ࣉ࣮ࣝ⾑₢ཬࡧࣉ࣮ࣝ⫢࣍ࣔࢪࢿ࣮ࢺࢧࣥࣉࣝࢆㄪ〇ࡋࡓ㸦n=3Ѝ1㸧ࠋࣉ࣮ࣝ⾑₢ཬࡧࣉ࣮ࣝ⫢࣍ࣔࢪࢿ࣮ࢺࢧࣥࣉࣝ࡟➼㔞ࡢ࢔ࢭࢺࢽࢺࣜࣝࢆῧຍࡋ㸪4°C㸪3000 rpm ࡛ 10 ศ㛫㐲ᚰศ㞳ࡋࡓࠋୖΎࢆ⭷ࣇ࢕ࣝࢱ࣮ࢆ⏝࠸࡚㐲ᚰℐ㐣ᚋ㸪ℐᾮࢆ HPLC㸦Agilent 1290 Infinity㸪
Agilent Technologies㸧࡟ UV ᳨ฟჾ࡜ Orbitrap ㉁㔞ศᯒ⿦⨨㸦Orbitrap Velos Pro㸪Thermo
Fisher Scientific 㸧ࢆ᥋ ⥆ࡋࡓ ᾮయ ࢡ ࣟ ࣐ࢺ ࢢ ࣛ ࣇ ࢕ ࣮ ࢱࣥ ࢹ ࣒ ㉁㔞ศ ᯒ ⿦ ⨨㸦 liquid
chromatography-tandem mass spectrometry coupled with UV detector㸪LC-UV/MS㸧࡟ὀධࡋ㸪24

Ἴ㛗 210nm ࡟࠾ࡅࡿ྾ගᗘཬࡧ positive ion mode ࡟࡚ᚓࡽࢀࡓ FBM ཬࡧ௦ㅰ≀ࡢ⢭ᐦ㉁㔞࡜ MS/MS ࣇࣛࢢ࣓ࣥࢺࡢ᝟ሗࢆࡶ࡜࡟ᵓ㐀ࢆ᥎ᐃࡋࡓࠋศᯒ࣒࢝ࣛࡣ CAPCELL PAK
C18 AQ㸦ȝm㸪3 × 250 mm㸪㈨⏕ᇽ㸧ࢆ⏝࠸ࡓࠋ⛣ື┦࡟ࡣ㸪࣑ࣜ Q Ỉ㸸ࢠ㓟㸦1000㸸1㸪
v/v㸧
㸦⛣ື┦ A㸧࡜࢔ࢭࢺࢽࢺࣜࣝ㸸ࢠ㓟㸦1000㸸1㸪v/v㸧
㸦⛣ື┦ B㸧ࢆ⏝࠸ࡓࠋ⛣ື┦ࡢ໙㓄ࡣ㸪ὀධᚋ 0 min ࠿ࡽ 10 min ࡲ࡛⛣ື┦ B ࡢ๭ྜࢆ 0%࡟ಖࡕ㸪10 min ࠿ࡽ 95 min ࡲ࡛ 0%࠿ࡽ 95%ࡲ࡛┤⥺ⓗ࡟ୖ᪼ࡉࡏ㸪95 min ࠿ࡽ 100 min ࡲ࡛ 100%࡜୍ᐃ࡜ࡋࡓࠋᮏ
ᐃ࡟࠾࠸࡚ FBM ཬࡧྛ௦ㅰ≀ࡢ᳨ฟ࡟⏝࠸ࡓࣉࣟࢺࣥ௜ຍయ[M + H]+ࡢ m/z ࢆ Table 2 ࡟♧ࡍࠋTable 2. List of m/z used in metabolite profiling study.
Analytes
FBM
CBMA
CCMF
2OH-FBM
pOH-FBM
MCF
MW441
CPPA
MCF-gluc
Nac-OH
Nac-acid

Ion mode

m/z
239.10263
194.08117
194.08117
255.09755
255.09755
196.09682
442.16425
210.07608
372.12891
298.11076
312.09002

PositiveII-2-iv㸧 MSI ࢆ⏝࠸ࡓ GSH ࡢኚືゎᯒ FBM ࢆ 0.5%࣓ࢳࣝࢭ࣮ࣝࣟࢫ࡟ᠱ⃮ࡋ࡚ 600 mg/10 mL/kg ࡛ PXB ࣐࢘ࢫ࡟⤒ཱྀᢞ୚ࡋ㸪24 ᫬㛫ᚋࡢ⫢⮚ࢆ᥇ྲྀࡋࡓࠋ⫢⮚ࢆࢻࣛ࢖࢔࢖ࢫ࡛෾⤖ࡋ㸪ࢡࣛ࢖࣑࢜ࢡࣟࢺ࣮࣒㸦CRYO
STAR NX70㸪Thermo Fisher Scientific㸧࡟࡚ 1ȝm ࡢཌࡉ࡟ⷧษࡋ࡚෾⤖⫢ษ∦ࢆసᡂࡋࡓࠋ
࣐ࢺࣜࢵࢡࢫᨭ᥼࣮ࣞࢨ࣮⬺㞳࢖࢜ࣥ໬㸦matrix assisted laser desorption/ionization㸪MALDI㸧ࡢ࣐ࢺࣜࢵࢡࢫ࡜ࡋ࡚ DHB ࢆ 0.1%ࢺࣜࣇࣝ࢜ࣟ㓑㓟ྵ᭷ 50%࣓ࢱࣀ࣮ࣝ⁐ᾮ࡟⁐ゎࡋ࡚෾⤖⫢ษ∦ୖ࡟ሬᕸࡋ㸪MSI ⏝ࡢศᯒࢧࣥࣉࣝ࡜ࡋࡓࠋ࣐ࢺࣜࢵࢡࢫሬᕸᚋࡢ෾⤖⫢ษ∦ࢆ Solarix XR㸦Bruker Daltonics㸧࡟࡚ศᯒࡋ㸪FBM㸪FBM ௦ㅰ≀ཬࡧෆᅉᛶ≀㉁ࡢศᕸࢆ
25

ゎᯒࢯࣇࢺ flexImaging㸦Bruker Daltonics㸧࡟࡚⏬ീ໬ࡋࡓࠋMSI ศᯒ࡟࠾࠸࡚

ᐃᑐ㇟ࡢ᳨ฟ࡟⏝࠸ࡓ࣒࢝ࣜ࢘࢖࢜ࣥ௜ຍయ[M + K]+ࡢ m/z ࢆ Table 3 ࡟♧ࡍࠋ
Table 3. List of m/z used in MSI.
Analytes
FBM
2-pp
Atropic acid
MW175
CPPA
Oxazine-dione
CBMA
CCMF
2OH-FBM
pOH-FBM
MCF
MW295
MCF-gluc
MW439
Nac-OH
Nac-acid
MW441
GSH
GSSG
Ophthalmic acidIon mode

m/z
277.0585
171.0207
187.0156
214.0265248.0320230.0214232.0370
232.0370
293.0534
293.0534
234.0527
334.0510
410.0848
478.1045
336.0666
350.0459
480.1201
346.0470
651.1151
328.0905

PositiveII-2-v㸧 FBM ࢆᢞ୚ᚋࡢ⫢ᶵ⬟ࣃ࣓࣮ࣛࢱ࣮ホ౯ II-2-iv ࡟࠾࠸࡚ FBM ࢆᢞ୚ࡋࡓ PXB ࣐࢘ࢫࡼࡾ⾑₢ࢆ᥇ྲྀࡋࡓࠋᚓࡽࢀࡓ⾑₢ࢆ⮫ᗋ໬Ꮫศᯒ⿦⨨㸦FUJI DRI-CHEM analyzer㸪ᐩኈࣇ࢖࣒ࣝ㸧࡛ศᯒࡋ㸪alanine aminotransferase
㸦ALT㸧㸪aspartate aminotransferase㸦AST㸧ཬࡧ alkaline phosphatase㸦ALP㸧ࢆᐃࡋࡓࠋ

II-2-vi㸧 ⤫ィゎᯒ

 FBM ࢆᢞ୚ᚋࡢ⫢ᶵ⬟ࣃ࣓࣮ࣛࢱ࣮ホ౯㸦II-2-v㸧࡟࠾࠸࡚㸪᭷ពᕪࡢ᳨ᐃࡣ t ᳨ᐃ࡟ࡼ26

ࡾᐇ᪋ࡋࡓࠋp ್ࡀ 0.05 ᮍ‶ࢆ⤫ィⓗ࡟᭷ព࡜ࡳ࡞ࡋࡓࠋ

27

➨ 3 ⠇ ⤖ᯝ

II-3-i㸧 PXB ࣐࢘ࢫ࡟࠾ࡅࡿ FBM ࡢ௦ㅰ≀ࣉࣟࣇ࢓࢖ࣝホ౯

FBM ࢆ PXB ࣐࢘ࢫ࡟ᢞ୚ᚋࡢ⾑₢ࢆ LC-UV/MS ศᯒࡋ㸪FBM ࡢ௦ㅰ≀ࣉࣟࣇ࢓࢖ࣝࢆホ౯ࡋࡓࠋ
FBM ࢆᢞ୚ᚋࡢ UV ࢡ࣐ࣟࢺࢢ࣒ࣛࡼࡾ㸪⾑₢୰࠿ࡽࡣ FBM ཬࡧ௦ㅰ≀࡜ࡋ࡚ OHFBM㸪CBMA㸪CCMF㸪MCF㸪Nac-OH ࡀ᳨ฟࡉࢀࡓ㸦Fig. 11㸦A㸧㸧ࠋࡲࡓ㸪ᢳฟ࢖࢜ࣥࢡ
࣐ࣟࢺࢢ࣒ࣛࡼࡾ㸪ࡇࢀࡽࡢ௦ㅰ≀࡟ຍ࠼ MW441㸪CPPA㸪MCF-gluc㸪Nac-acid ࡀ᳨ฟࡉࢀࡓ㸦Fig. 11㸦B㸧㸧ࠋ(A)UV Chromatogram

28

(BB) Extracted Ion Chromatogram

Dosed

FBM
(m/z = 239.10263)

Control

Dosed

CBMA or CCMF
(m/z = 194.08117)

Control

2OH-FBM or

Dosed

pOH FBM(m/z = 255.09755)

Control29

DosedMCF(m/z = 196.09682)

Control

MW441

Dosed

(m/z = 442.16425)

Control

Dosed

CPPA
(m/z = 210.07608)

Control

30

DosedMCF-gluc(m/z = 372.12891)

Control

Nac-OH

Dosed(m/z = 298.11076)

Control

Dosed

Nac-acid(m/z = 312.09002)

Control

Fig. 11. UV (A) and extracted ion chromatograms (B) of plasma from PXB--mice.
FBM were orally administered to PXB-mice at 600 mg/kg and plasma samples were colledted
2 h after administration. Trace: detected only by MS.

31ḟ࡟ FBM ࢆ PXB ࣐࢘ࢫ࡟ᢞ୚ᚋࡢ⫢࣍ࣔࢪࢿ࣮ࢺࢆ LC-UV/MS ศᯒࡋࡓ࡜ࡇࢁ㸪UVࢡ࣐ࣟࢺࢢ࣒ࣛࡼࡾ㸪FBM ࡀ᳨ฟࡉࢀࡓ㸦Fig. 12㸦A㸧㸧ࠋࡲࡓ㸪ᢳฟ࢖࢜ࣥࢡ࣐ࣟࢺࢢࣛ
࣒ࡼࡾ㸪FBM ࡟ຍ࠼࡚௦ㅰ≀࡜ࡋ࡚ CBMA㸪CCMF㸪OH-FBM㸪MCF㸪MW441㸪CPPA ࡀ᳨ฟࡉࢀࡓ㸦Fig. 12㸦B㸧㸧ࠋ(AA)UUV Chromatogram
2OH-FBM or pOH-FBM(trace)CPPA
MW441 (trace)
CBMA or CCMF (trace)(trace)FBM

MCF(trace)
Dosed

Control(BB) Extracted Ion Chromatogram
Dosed

FBM
(m/z = 239.10263)

Control

32

CBMA or CCMF

Dosed

(m/z = 194.08117)

Control

2OH-FBM or

Dosed

p-OH FBM(m/z = 255.09755)

Control

Dosed

MCF(m/z = 196.09682)

Control33

MW441

Dosed(m/z = 442.16425)

Control

CPPA

Dosed

(m/z = 210.07608)

Control

Fig. 12. UV (A) and extracted ion chromatograms (B) of liver homogenete from PXB--mice.
FBM were orally administered to PXB-mice at 600 mg/kg and liver samples were colledted
2 h after administration. Trace: detected only by MS.

FBM ࢆ PXB ࣐࢘ࢫ࡟ᢞ୚ᚋࡢ⾑₢ཬࡧ⫢࣍ࣔࢪࢿ࣮ࢺࡢ LC-UV/MS ศᯒ࡟᳨࡚ฟࡉࢀ
ࡓ FBM ࡜௦ㅰ≀ࡢࣆ࣮ࢡ㠃✚್ࢆ Table 4 ࡟♧ࡍࠋ34

Table 4.. Comparison of UV peak area of FBM and its metabolites.
(A) Plasma
CBMA
or
CCMF

CBMA
or
CCMF

2OH-FBM 2OH-FBM
oror
pOH-FBM pOH-FBM

MCF% of FBM

0.28

0.14

0.50

1.51

Nac-OH

Nacacid

CPPA

Trace

Trace

1.30

-

-

100

-

1.78

-

MCF

MW441

CPPA

FBM

MCFgluc

Nac-OH

Nacacid

N.D.

N.D.

N.D.

-

-

-

UV Peak
329052 161698 596233 1789046 1538782
Area

FBM

MCFgluc

MW441

118625182 Trace 2115013 Trace(B) Liver
CBMA
or
CCMF

CBMA
or
CCMF

2OH-FBM 2OH-FBM
oror
pOH-FBM pOH-FBMUV Peak
Area

Trace

Trace

Trace

Trace

Trace

Trace

Trace

% of FBM

-

-

-

-

-

-

-

100

FBM were orally administered to PXB--mice at 600 mg/kg and pllasma (A) and liver (B) samples
were colledted 2 h after administration. Trace: detected only by MS.. N.D.: not deteted.

FBM ࢆᢞ୚ᚋࡢ PXB ࣐࢘ࢫ⾑₢୰࠿ࡽ᳨ฟࡉࢀࡓ௦ㅰ≀ࡢ UV ࣆ࣮ࢡ㠃✚ࢆẚ㍑ࡍࡿ
࡜㸪᳨ฟࡉࢀࡓ࠸ࡎࢀࡢ௦ㅰ≀ࡶࡑࡢ๭ྜࡣ FBM ࡢ 2%௨ୗ࡛࠶ࡗࡓࠋࡲࡓ㸪⫢࣍ࣔࢪࢿ࣮ࢺ࡟࠾࠸࡚㸪௦ㅰ≀ࡣ MS ࡟ࡼࡿᢳฟ࢖࢜ࣥࢡ࣐ࣟࢺࢢ࣒࡛ࣛࡢࡳ᳨ฟࡉࢀ㸪ࡑࡢ⏕ᡂ㔞ࡣࡈࡃࢃࡎ࠿࡛࠶ࡿࡶࡢ࡜⪃࠼ࡽࢀࡓ㸦Table 4㸧ࠋ
PXB ࣐࢘ࢫࡢ⾑₢ཬࡧ⫢࣍ࣔࢪࢿ࣮ࢺ୰࠿ࡽ᳨ฟࡉࢀࡓ௦ㅰ≀ࡢ࠺ࡕ㸪OH-FBM ࡣ FBM
ࡢ㓟໬௦ㅰ࡟ࡼࡾ⏕ᡂࡍࡿ௦ㅰ≀㸪MCF ࡣ FBM ࡢຍỈศゎ௦ㅰ࡟ࡼࡾ⏕ᡂࡍࡿ௦ㅰ≀࡛࠶ࡾ㸪ࡲࡓ CBMA㸪CCMF㸪CPPA㸪MCF-gluc ࡣ MCF ࠿ࡽࡢ㏲ḟ௦ㅰ࡟ࡼࡾ⏕ᡂࡍࡿ௦ㅰ
≀࡛࠶ࡿ࡜᥎ᐃࡉࢀࡓ㸦Fig. 3㸧ࠋࡉࡽ࡟㸪MW441 ࡣ MCF ࡼࡾ⏕ᡂࡍࡿ཯ᛂᛶ௦ㅰ≀ 2-PP
ࡀ GSH ᢪྜࢆཷࡅ࡚⏕ᡂࡍࡿ௦ㅰ≀࡛࠶ࡾ㸪Nac-OH ཬࡧ Nac-acid ࡣ GSH ᢪྜయࡀࡉࡽ
࡟௦ㅰࡉࢀ࡚⏕ᡂࡍࡿ N-࢔ࢭࢳࣝࢩࢫࢸ࢖ࣥᢪྜయ࡛࠶ࡿ࡜᥎ᐃࡉࢀࡓ㸦Fig. 3㸧ࠋࡇࢀࡽࡢ⤖ᯝࡼࡾ㸪
FBM ࢆ PXB ࣐࢘ࢫ࡟ᢞ୚ᚋ㸪
PXB ࣐࢘ࢫ୰ࡢࣄࢺ⫢⮚࡟࠾࠸࡚ FBM
ࡢ㓟໬ཬࡧຍỈศゎࡀ㉳ࡇࡾ㸪ࡉࡽ࡟ MCF ࢆ௓ࡋ࡚⏕ᡂࡍࡿ 2-PP ࡀ GSH ᢪྜ࡟ࡼࡿゎẘ௦ㅰࢆཷࡅࡿࡶࡢ࡜⪃࠼ࡽࢀࡓࠋ
35

II-3-ii㸧 MSI ࢆ⏝࠸ࡓ GSH ࡢኚືゎᯒ

II-3-i ࡟࠾࠸࡚㸪FBM ࢆ PXB ࣐࢘ࢫ࡟ᢞ୚ᚋࡢ⾑₢ཬࡧ⫢࣍ࣔࢪࢿ࣮ࢺ࠿ࡽ㸪2-PP ࡀ
GSH ᢪྜࢆཷࡅ࡚⏕ᡂࡍࡿ௦ㅰ≀ࡀ」ᩘ᳨ฟࡉࢀࡓࡇ࡜࠿ࡽ㸪FBM ࡢᢞ୚ࡀࣄࢺࡢ⫢⮚࡟୚࠼ࡿᙳ㡪࡟ࡘ࠸࡚ゎᯒࡍࡿࡓࡵ㸪PXB ࣐࢘ࢫ࡟ FBM ࢆᢞ୚ᚋࡢ⫢⮚࡟࠾ࡅࡿ GSH ࡢኚື࡟ࡘ࠸࡚ゎᯒࢆ⾜ࡗࡓࠋ
FBM ࢆᢞ୚ᚋࡢ PXB ࣐࢘ࢫ⫢⮚ࢆ MSI ศᯒࡋ㸪FBM ཬࡧ GSH ࡢศᕸࢆྍど໬ࡋࡓ࡜ࡇࢁ㸪FBM ࡢᢞ୚࡟ࡼࡾ GSH ࡀῶᑡഴྥࢆ♧ࡍࡇ࡜ࡀ☜ㄆࡉࢀࡓ㸦Fig. 13㸧
ࠋ

Dosed

Control

Dosed
Blank
(slide glass)
FBM

GSH

Fig. 13. Visualization of FBM and GSH on liver sections from PXB--mice.
FBM were orally administered to PXB-mice at 600 mg/kg and liver samples were colledted
24 h after administration. Distribution of FBM and GSH were analyzed by MSI.

36

FBM ࡢᢞ୚ࡀࣄࢺࡢ⫢⮚࡟ཬࡰࡍᙳ㡪࡟ࡘ࠸࡚ࡉࡽ࡟ゎᯒࡍࡿࡓࡵ㸪FBM ࢆᢞ୚ᚋࡢ
FBM ௦ㅰ≀㸪ࡲࡓෆᅉᛶ≀㉁࡜ࡋ࡚㓟໬ᆺࢢࣝࢱࢳ࢜ࣥ㸦GSSG㸧㸪ࡉࡽ࡟ෆᅉᛶ GSH ࡀῶᑡࡋ㸪GSH ྜᡂࡀάᛶ໬ࡍࡿ㐣⛬࡛ቑຍࡍࡿࡇ࡜࠿ࡽ㸪GSH ᯤῬࡢ࣐࣮࣮࢝࡜ࡋ࡚▱ࡽࢀࡿ࢜ࣇࢱ࣑ࣝࣥ㓟ࡢኚື࡟ࡘ࠸࡚ゎᯒࢆ⾜ࡗࡓࠋࡋ࠿ࡋ࡞ࡀࡽ㸪᳨ウࢆ⾜ࡗࡓࡇࢀࡽࡢ≀㉁࡟㛵ࡋ࡚ࡣ MSI ศᯒ࡛ࡣ᳨ฟࡍࡿࡇ࡜ࡀ࡛ࡁ
࡞࠿ࡗࡓࠋ㸦Fig. 14㸧
Atropic acid (N.D.)

Dosed

Control

Dosed
Blank
(slide glass)
2-PP (N.D.)

MW175 (N.D.)

CPPA (N.D.)

Oxazine-dione (N.D.)

CBMA or CCMF (N.D.)

OH-FBM (N.D.)

MCF (N.D.)

MW295 (N.D.)

MCF-gluc (N.D.)

MW439 (N.D.)

37

Nac-OH (N.D.)

Nac-acid (N.D.)

MW441(N.D.)

GSSG (N.D.)

Ophthalmic acid (N.D.)

Fig. 14. Visualization of FBM metabolites, GSSG and ophthalmic acid on liver sections from
PXB-mice.
FBM were orally administered to PXB-mice at 600 mg/kg and liver samples were colledted 24 hafter administration. Distribution of FBM metabolites, GSSG and ophthalmic acid wereanalyzed by MSI. N.D.: not detected.

38

II-3-iii㸧 FBM ࢆᢞ୚ᚋࡢ⫢ᶵ⬟ࣃ࣓࣮ࣛࢱ࣮ホ౯

II-3-ii ࡟࡚ FBM ࢆᢞ୚ࡋࡓ PXB ࣐࢘ࢫࡢ⾑₢ࢆ⏝࠸࡚㸪FBM ࢆᢞ୚ᚋࡢ⫢ᶵ⬟ホ౯ࢆ⾜ࡗࡓࠋ⾑₢୰ࡢ ALT㸪AST ཬࡧ ALP ࢆᐃࡋࡓ࡜ࡇࢁ㸪࠸ࡎࢀࡢࣃ࣓࣮ࣛࢱ࣮ࡶࢥࣥࢺ࣮ࣟࣝ

⩌࡜ẚ㍑ࡋ࡚᭷ព࡞ᕪࡣㄆࡵࡽࢀ࡞࠿ࡗࡓ㸦Fig. 15㸧
ࠋ
ALP

AST
500

800

200

400

600

150

300

100
50

U/L

250

U/L

U/L

ALT

200

100

0

0

0

Control FBM

400

200

Control FBM

Control FBM

Fig. 15. Plasma ALT, AST and ALP levels after administration of FBM.
FBM were orally administered to PXB-mice at 600 mg/kg and plasma samples werecolledted 24 h after administration. Data represent the mean ± SD (n=3).

39➨ 4 ⠇ ᑠᣓᮏ❶࡛ࡣ㸪PXB ࣐࢘ࢫࢆ⏝࠸࡚ FBM ࢆᢞ୚ᚋࡢ௦ㅰ≀ࣉࣟࣇ࢓࢖ࣝࢆホ౯ࡍࡿ࡜࡜ࡶ
࡟㸪 FBM ࡢᢞ୚ࡀࣄࢺࡢ⫢⮚࡟୚࠼ࡿᙳ㡪࡟ࡘ࠸᳨࡚ウࢆ⾜ࡗࡓࠋ

PXB ࣐࢘ࢫࢆ⏝࠸ࡓ FBM ࡢ௦ㅰ≀ࣉࣟࣇ࢓࢖ࣝホ౯࡟࠾࠸࡚㸪⾑₢୰࠿ࡽࡣᮍኚ໬య
ཬࡧ 10 ✀㢮ࡢ௦ㅰ≀ࡀ᳨ฟࡉࢀ㸦Fig. 11㸧㸪⫢࣍ࣔࢪࢿ࣮ࢺ୰࠿ࡽࡣᮍኚ໬యཬࡧ 7 ✀㢮ࡢ௦ㅰ≀ࡀ᳨ฟࡉࢀࡓ㸦Fig.12㸧ࠋࡇࢀࡽࡢ௦ㅰ≀࡟ࡣ㸪FBM ࡢ㓟໬௦ㅰ≀࡛࠶ࡿ OH-FBM㸪ຍỈศゎ௦ㅰ≀࡛࠶ࡿ MCF ࡀྵࡲࢀࡿ࡜࡜ࡶ࡟㸪
MCF ࡢ㏲ḟ௦ㅰ࡟ࡼࡾ⏕ᡂࡍࡿ CBMA㸪
CCMF㸪CPPA㸪MCF-gluc ࡟ຍ࠼࡚㸪
2-PP ࡀ GSH ᢪྜࢆཷࡅ࡚⏕ᡂࡍࡿ௦ㅰ≀࡛࠶ࡿ MW441㸪
GSH ᢪྜయࡀࡉࡽ࡟௦ㅰࡉࢀ࡚⏕ᡂࡍࡿ N-࢔ࢭࢳࣝࢩࢫࢸ࢖ࣥᢪྜయ࡛࠶ࡿ Nac-OH ཬࡧ
Nac-acid ࡀྵࡲࢀ࡚࠸ࡓࠋ ᮏ◊✲࡛ࡣ㸪࣐࢘ࢫࡸࣛࢵࢺࢆ⏝࠸ࡓ௦ㅰ≀ࣉࣟࣇ࢓࢖ࣝࡢ᳨ウࡣ⾜ࡗ࡚࠸࡞࠸ࡓࡵ㸪
PXB ࣐࢘ࢫ࡜ᐇ㦂ື≀ࡢ௦ㅰ≀ࣉࣟࣇ࢓࢖ࣝࡢ✀ᕪ࡟ࡘ࠸࡚ࡣ༑ศ࡟ࡣ᳨ド࡛ࡁ࡚࠸࡞࠸ࠋ
ᚑࡗ࡚㸪✀ᕪ࡟㛵ࡋ࡚ࡣ௒ᚋ᭦࡞ࡿ᳨ウࡀᚲせ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࡶࡢࡢ㸪PXB ࣐࢘ࢫࢆ⏝࠸ࡓࡇࢀࡲ࡛ࡢ◊✲࡟࠾࠸࡚㸪ࣄࢺ≉␗ⓗ௦ㅰ≀ࢆ᳨ฟྍ⬟࡛࠶ࡗࡓࡇ࡜ࡀከᩘሗ࿌ࡉ
ࢀ࡚࠸ࡿ㸦De Serres et al.㸪2011㸹Inoue et al.㸪2009㸹Kamimura et al.㸪2010㸹Yamazaki et al.㸪2010㸧ࠋࡲࡓ㸪ࣄࢺ࡟࠾࠸࡚㸪FBM ࢆᢞ୚ᚋ࡟ᒀ୰࡟᤼ἥࡉࢀࡿ 2OH-FBM ࡜ pOH-FBM ࡢ࿴ࡣᢞ୚㔞ࡢ 10%㸪Nac-OH ࡜ Nac-acid ࡢ࿴ࡣᢞ୚㔞ࡢ 6.3%࡛࠶ࡿࡢ࡟ᑐࡋ㸪ࣛࢵࢺ࡟࠾
࠸࡚ࡣ OH-FBM ࡜ pOH-FBM ࡢ࿴ࡀ 62%࡜㠀ᖖ࡟㧗ࡃ㸪Nac-OH ࡜ Nac-acid ࡢ࿴ࡀ 1.1%࡜
ప࠸ࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ㸦Dieckhaus et al.㸪2000㸧ࠋᮏ◊✲࡟࠾࠸࡚㸪⾑₢୰࠿ࡽ᳨ฟࡉ
ࢀࡓ OH-FBM ࡜ pOH-FBM ࡢ࿴ࡣ FBM ࡟ᑐࡋ࡚ 2.01%㸪Nac-OH ࡜ Nac-acid ࡢ࿴ࡣ FBM
࡟ᑐࡋ࡚ 1.78%࡜ྠ⛬ᗘ࡛࠶ࡗࡓࡇ࡜ࢆ㋃ࡲ࠼ࡿ࡜㸦Table 4㸦A㸧㸧
㸪PXB ࣐࢘ࢫ࡟࠾ࡅࡿ
FBM ࡢ௦ㅰ≀ࣉࣟࣇ࢓࢖ࣝࡣ㸪ࣄࢺ࡟㏆࠸ࡶࡢ࡛࠶ࡗࡓ࡜᝿ᐃࡉࢀࡿࠋ
ᮏ◊✲࡛ࡣ㸪PXB ࣐࢘ࢫࢆ⏝࠸ࡓ in vivo ヨ㦂࡟࠾࠸࡚ࡶ㸪཯ᛂᛶ௦ㅰ≀࡛࠶ࡿ 2-PP ࡣ

40᳨ฟࡉࢀ࡞࠿ࡗࡓ㸦Fig. 11㸪Fig. 12㸧ࠋ୍᪉࡛㸪2-PP ࡀ GSH ᢪྜࢆཷࡅ࡚⏕ᡂࡍࡿ௦ㅰ≀ࡀ」ᩘ᳨ฟࡉࢀࡓࠋ2-PP ࡢ཯ᛂᛶࡀ㧗࠸ࡇ࡜ࢆ㋃ࡲ࠼ࡿ࡜㸦Dieckhaus et al.㸪2001㸹Thompson
et al.㸪1996㸧
㸪2-PP ࡣ⏕ᡂᚋ࡟㏿ࡸ࠿࡟ GSH ᢪྜ࡟ࡼࡿゎẘ௦ㅰࢆཷࡅࡿࡶࡢ࡜⪃࠼ࡽࢀ
ࡓࠋ

MSI ࢆ⏝࠸ࡓゎᯒ࡟ࡼࡾ㸪FBM ࢆᢞ୚ᚋ㸪PXB ࣐࢘ࢫࡢ⫢⮚࡟࠾࠸࡚㸪ෆᅉᛶ GSH ࡀῶᑡഴྥࢆ♧ࡍࡇ࡜ࡀ☜ㄆࡉࢀࡓ㸦Fig. 13㸧
ࠋࡇࡢ⤖ᯝࡼࡾ㸪FBM ࡟ࡼࡿ⫢ẘᛶ࡟ࡣ 2-PPࢆゎẘ௦ㅰࡍࡿ㐣⛬࡛ GSH ࡀῶᑡࡍࡿࡇ࡜ࡀ㛵୚ࡋ࡚࠸ࡿࡶࡢ࡜⪃࠼ࡽࢀࡓࠋ
FBM ࡢᢞ୚ࡀ⫢⮚࡟ཬࡰࡍᙳ㡪࡟ࡘ࠸࡚ࡉࡽ࡟ゎᯒࡍࡿࡓࡵ㸪FBM ௦ㅰ≀ཬࡧ GSSG
ࡸࢢࣝࢱࢳ࢜ࣥᯤῬ࣐࣮࣮࡛࢝࠶ࡿ࢜ࣇࢱ࣑ࣝࣥ㓟ࡢ᳨ฟࢆヨࡳࡓ࡜ࡇࢁ㸪࠸ࡎࢀࡢ≀㉁ࡶ᳨ฟࡉࢀ࡞࠿ࡗࡓ㸦Fig. 14㸧ࠋMSI ࡢ࢖࢜ࣥ໬ἲ࡜ࡋ୍࡚⯡ⓗ࡟⏝࠸ࡽࢀ࡚࠸ࡿ MALDIࡣ㸪LC-MS ࡛ỗ⏝ࡉࢀࡿ࢖࢜ࣥ໬ἲ࡛࠶ࡿ࢚ࣞࢡࢺࣟࢫࣉ࣮ࣞ࢖࢜ࣥ໬ἲ㸦electrosprayionization, ESI㸧࡜ẚ㍑ࡋ࡚㸪పศᏊ໬ྜ≀ࡢ᳨ฟឤᗘࡣప࠸ഴྥ࡟࠶ࡿࠋࡲࡓ㸪PXB ࣐࢘
ࢫࡢ⫢࣍ࣔࢪࢿ࣮ࢺࢆ⏝࠸ࡓ LC-UV/MS ศᯒ࡟࠾࠸࡚㸪FBM ௦ㅰ≀ࡢ㔞ࡣᮍኚ໬య࡜ẚ࡭࡚ࡈࡃࢃࡎ࠿࡛࠶ࡿࡇ࡜ࡀ☜ㄆࡉࢀ࡚࠸ࡿ㸦Table 4㸧ࠋࡇ࠺ࡋࡓⅬࢆ⪃៖ࡍࡿ࡜㸪ᮏ◊✲࡛ࡣ MSI ศᯒ࡟࠾࠸࡚㸪FBM ௦ㅰ≀ࡸࡇࢀࡽࡢෆᅉᛶ≀㉁ࢆ᳨ฟࡍࡿࡢ࡟༑ศ࡞ឤᗘࢆᚓࡽࢀ࡞࠿ࡗࡓࡶࡢ࡜⪃࠼ࡽࢀࡿࠋ ⫢⮚࡟࠾ࡅࡿ GSH ࡢῶᑡ࡟ࡣ㸪2-PP ࡢᢪྜ௦ㅰ࡟ࡼࡿῶᑡ࡟ຍ࠼࡚㸪㓟໬ࢫࢺࣞࢫࡢ
㑏ඖ࡟ࡼࡾ GSH ࡀ GSSG ࡟㓟໬ࡉࢀࡓࡇ࡜࡟ࡼࡿῶᑡࡀ㛵୚ࡋ࡚࠸ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋᮏ◊✲࡟࠾࠸࡚ࡣ㸪㓟໬ࢫࢺࣞࢫࡢ㑏ඖ࡟ࡼࡗ࡚ቑຍࡍࡿ GSSG ࡢኚ໬ࢆゎᯒ࡛ࡁ࡚࡞࠸ࡓࡵ㸪FBM ࡢᢞ୚ᚋ࡟ PXB ࣐࢘ࢫࡢ⫢⮚࡛ぢࡽࢀࡓ GSH ࡢῶᑡ࡟㛵ࡋ࡚㸪2-PP ࡢᢪྜ௦ㅰ࡜㸪㓟໬ࢫࢺࣞࢫࡢ㑏ඖ࡟ࡼࡿᙳ㡪ࢆศ㞳ホ౯࡛ࡁ࡞࠸ࡀ㸪ᑡ࡞ࡃ࡜ࡶ㸪FBM ࡢᢞ୚࡟ࡼࡗ࡚⏕ࡌࡿ GSH ࡢῶᑡࡀ⫢ẘᛶࡢⓎ⌧࡟㛵୚ࡋ࡚࠸ࡿࡶࡢ࡜⪃࠼ࡽࢀࡓࠋ ᮏ◊✲࡛ࡣ㸪PXB ࣐࢘ࢫࡢ⫢⮚ࢆ MSI ศᯒࡍࡿࡇ࡜࡛㸪FBM ࡢᢞ୚࡟ࡼࡾ⫢⮚࡟࠾࠸41

࡚ GSH ࡀῶᑡࡍࡿࡇ࡜ࢆ┤᥋ⓗ࡟ホ౯ࡍࡿࡇ࡜ࡀྍ⬟࡛࠶ࡗࡓࠋ⫢ẘᛶࡢ࣓࢝ࢽࢬ࣒ࢆゎ᫂ࡍࡿ࡟࠶ࡓࡾ㸪⮫ᗋ࡛ࣄࢺࡢ⫢⮚ࢆ⏝࠸࡚ GSH ࡢኚືࢆ┤᥋ホ౯ࡍࡿࡇ࡜ࡣᅔ㞴࡛࠶ࡿࡇ࡜࠿ࡽ㸪PXB ࣐࢘ࢫࡣ⸆≀࡟ࡼࡿ⫢ẘᛶࡢ࣓࢝ࢽࢬ࣒ࢆゎ᫂ࡍࡿୖ࡛᭷⏝࡞ࣄࢺ໬ື≀ࣔࢹࣝ࡟࡞ࡿࡶࡢ࡜⪃࠼ࡽࢀࡓࠋ
ࡲࡓ㸪ᮏ◊✲࡛ࡣ࣐࢘ࢫ⫢⣽⬊ࡢ኱㒊ศࡀࣄࢺ⫢⣽⬊࡟⨨᥮ࡉࢀࡓ㸪㧗⨨᥮ PXB ࣐࢘ࢫࢆ⏝࠸᳨࡚ウࢆ⾜ࡗࡓࠋSanoh ࡽ㸦2017㸧ࡣ㸪ࣄࢺ⫢⣽⬊ࡢ⨨᥮⋡ࡀ୰⛬ᗘࡢ PXB ࣐࢘ࢫࢆ⏝࠸࡚㸪amiodarone ࢆᢞ୚ᚋ࡟ෆᅉᛶࣜࣥ⬡㉁ࢆ MSI ศᯒࡋ㸪࣐࢘ࢫ࡜ࣄࢺ࡟࠾ࡅࡿࣜࣥ⬡㉁ࡢ✀ᕪࢆゎᯒࡋࡓࡇ࡜ࢆሗ࿌ࡋ࡚࠸ࡿࠋ
௒ᚋ㸪ࡇࡢࡼ࠺࡟ࣄࢺ⫢⣽⬊ࡢ⨨᥮⋡ࡀ୰⛬ᗘࡢ PXB ࣐࢘ࢫࢆ⏝࠸ࡿࡇ࡜࡛㸪FBM ᢞ୚࡟ࡼࡿ GSH ኚືࡢ✀ᕪ࡟ࡘ࠸࡚ࡶ㸪ࡉࡽ࡟᳨ウࡍࡿࡇ࡜ࡀྍ⬟࡟࡞ࡿࡶࡢ࡜⪃࠼ࡽࢀࡿࠋ

FBM ࢆᢞ୚ᚋࡢ⫢ᶵ⬟ࣃ࣓࣮ࣛࢱ࣮ホ౯࡟࠾࠸࡚㸪ALT㸪AST㸪ALP ࡢ࠸ࡎࢀࡶࢥࣥࢺ
࣮ࣟࣝ⩌࡜ẚ㍑ࡋ࡚᭷ព࡞ᕪࡣㄆࡵࡽࢀ࡞࠿ࡗࡓ㸦Fig. 15㸧
ࠋMcGee ࡽ㸦1998㸧ࡣ㸪FBM ࢆ
3000-5000 mg/kg ࡛࣐࢘ࢫ࡟༢ᅇ⤒ཱྀᢞ୚ࡋࡓ㝿࡟㸪ẘᛶᡤぢࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࡇ࡜㸪ࡲࡓ㸪FBM ࢆ 300-600 mg/kg ࡛ࣛࢵࢺཬࡧ࢖ࢾ࡟཯᚟⤒ཱྀᢞ୚ࡋࡓ㝿࡟㸪ALT㸪AST㸪ALP ࡢ୍㐣ᛶࡢୖ᪼ࡀㄆࡵࡽࢀࡓࡶࡢࡢ㸪⫢⮚࡟࠾࠸࡚⤌⧊Ꮫⓗ࡞ኚ໬ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࡇ࡜
ࢆሗ࿌ࡋ࡚࠸ࡿࠋ
୍᪉࡛㸪Kakuni ࡽ㸦2012㸧ࡣ㸪ࣄࢺ࡟࠾࠸࡚཯ᛂᛶ௦ㅰ≀࡟㉳ᅉࡍࡿ IDT ࡟ࡼࡾ⫢ẘᛶ
ࢆⓎ⑕ࡍࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿ troglitazone ࢆ PXB ࣐࢘ࢫ࡟ 1000 mg/kg ࡛ 14 ᪥ཬࡧ 23 ᪥㛫཯᚟ᢞ୚ࡋࡓ࡜ࡇࢁ㸪ALT ཬࡧ AST ࡢୖ᪼ࡀぢࡽࢀࡓࡇ࡜ࢆሗ࿌ࡋ࡚࠸ࡿࠋ
ࡇ࠺ࡋࡓ᝟ሗࢆ㋃ࡲ࠼ࡿ࡜㸪PXB ࣐࢘ࢫࡣ IDT ࢆᘬࡁ㉳ࡇࡍ⸆๣࡛⫢ẘᛶࢆⓎ⑕ࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࡶࡢࡢ㸪ᮏ◊✲࡟࠾࠸࡚ࡣ㸪FBM ࢆᢞ୚ᚋ࡟ ALT㸪AST ཬࡧ ALP ࡢୖ᪼ࡀㄆࡵࡽࢀ࡞࠿ࡗࡓࡇ࡜࠿ࡽ㸪PXB ࣐࢘ࢫ࡟࠾࠸࡚ FBM ࡢᢞ୚࡟ࡼࡿ⫢ẘᛶࡣ㉳ࡇࡗ࡚࠸࡞࠸ࡶࡢ࡜⪃࠼ࡽࢀࡓࠋࡇࡢ⤖ᯝࡣ࣐࢘ࢫ㸪ࣛࢵࢺཬࡧ࢖ࢾࢆ⏝࠸ࡓ McGee ࡽ㸦1998㸧ࡢሗ࿌࡜୍⮴ࡍࡿࡶࡢ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋ

42ᚑࡗ࡚㸪཯ᛂᛶ௦ㅰ≀ࡢ⏕ᡂ࠿ࡽ⫢ẘᛶࡢⓎ⌧࡟⮳ࡿࡲ࡛ࡢ࣓࢝ࢽࢬ࣒ࢆࡉࡽ࡟ゎ᫂ࡍࡿࡓࡵ࡟ࡣ㸪FBM ࡢᢞ୚㔞ࢆୖࡆࡿ㸪࠶ࡿ࠸ࡣ཯᚟ᢞ୚ࢆࡍࡿ࡞࡝㸪௒ᚋࡉࡽ࡞ࡿ᳨ウࡀ
ᚲせ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋ
ࡉࡽ࡟㸪IDT ࡟ࡼࡿ⫢ẘᛶ࡟ࡣ㸪ච␿཯ᛂࡀ㛵୚ࡍࡿࡇ࡜ࡶሗ࿌ࡉࢀ࡚࠸ࡿ㸦Park et al.㸪
2005㸹Satoh et al.㸪1985㸹Walgren et al.㸪2005㸧ࠋPXB ࣐࢘ࢫࡣච␿୙඲ࡢᛶ㉁ࢆ᭷ࡍࡿࡇ࡜
࠿ࡽ㸪FBM ࡟ࡼࡿ⫢ẘᛶࡀච␿཯ᛂࢆ௓ࡋࡓࡶࡢ࡛࠶ࡿሙྜ㸪PXB ࣐࢘ࢫࢆ⏝࠸ࡓ᳨ウ࡛ࡣ㸪⫢ẘᛶࢆ᳨ฟ࡛ࡁ࡞࠸ྍ⬟ᛶࡶ⪃࠼ࡽࢀࡿࠋ
ᚑࡗ࡚㸪௒ᚋ㸪FBM ࡟ࡼࡿ⫢ẘᛶࡢ඲యീࢆ᫂ࡽ࠿࡟ࡋ࡚࠸ࡃࡓࡵ࡟ࡣ㸪཯ᛂᛶ௦ㅰ≀࡟㛵ࡍࡿ᳨ウ࡟ຍ࠼㸪ච␿⣔ࡢ㛵୚ࡸ㸪IDT ࡢ 3 ࡘࡢせᅉࡢ࠺ࡕࠕ㑇ఏⓗせᅉࠖࡸࠕ⎔ቃせᅉࠖ࡟ࡘ࠸࡚ࡶὀ┠ࡋ㸪ࡉࡽ࡟᳨ウࢆ⾜࠺ᚲせࡀ࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋ

43

⤖ㄽ

ᮏ◊✲࡛ࡣ㸪in vitro ௦ㅰヨ㦂⣔ཬࡧ PXB ࣐࢘ࢫࢆ⏝࠸࡚ FBM ࡢ཯ᛂᛶ௦ㅰ≀⏕ᡂ࣓࢝ࢽࢬ࣒࡟ࡘ࠸᳨࡚ウࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝ㸪௨ୗࡢ▱ぢࡀᚓࡽࢀࡓࠋ

ࣄࢺ CYP ཬࡧ CES Ⓨ⌧⣔࣑ࢡࣟࢯ࣮࣒ࢆ⏝࠸ࡓ᳨ウ࠿ࡽ㸪
FBM ࡢ㓟໬௦ㅰ࡟ CYP2C8㸪
CYP2C9 ཬࡧ CYP2E1 ࡀ㛵୚ࡋ㸪ຍỈศゎ௦ㅰ࡟ CES1c ࡀ㛵୚ࡍࡿࡶࡢ࡜⪃࠼ࡽࢀࡓࠋ
ࡇࢀࡽࡢ⤖ᯝࡼࡾ㸪CES1c ࢆ௓ࡋࡓຍỈศゎ௦ㅰࡀ 2-PP ࡢ⏕ᡂ࡟㛵୚ࡋ㸪CYP2C8㸪
CYP2C9 ཬࡧ CYP2E1 ࢆ௓ࡋࡓ㓟໬௦ㅰࡀ 2-PP ࡢ⏕ᡂࢆᅇ㑊ࡍࡿ㔜せ࡞௦ㅰ⤒㊰࡜࡞ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓ㸦Fig. 16㸧
ࠋ

FBM ࢆ PXB ࣐࢘ࢫ࡟ᢞ୚ᚋ㸪⾑₢ཬࡧ⫢⮚୰࠿ࡽ 2-PP ࡀ GSH ᢪྜࢆཷࡅ࡚⏕ᡂࡍࡿ௦ㅰ≀ࡀ」ᩘ᳨ฟࡉࢀࡓࠋࡲࡓ㸪FBM ࡢᢞ୚࡟ࡼࡾࠊ⫢⮚࡟࠾࠸࡚ෆᅉᛶ GSH ࡀపୗࡍࡿࡇ࡜ࡀ☜ㄆࡉࢀࡓࠋࡇࢀࡽࡢ⤖ᯝࡼࡾ㸪FBM ࡢᢞ୚࡟ࡼࡾ⏕ᡂࡍࡿ 2-PP ࢆゎẘ௦ㅰࡍࡿ㐣⛬࡛ GSH ࡀῶᑡࡍࡿࡇ࡜ࡀ FBM ࡟ࡼࡿ⫢ẘᛶ࡟㛵୚ࡋ࡚࠸ࡿࡶࡢ࡜⪃࠼ࡽࢀࡓ
㸦Fig.
16㸧ࠋ

PXB ࣐࢘ࢫࡣ㸪ࣄࢺࡢ௦ㅰ≀ࣉࣟࣇ࢓࢖ࣝ࡟ຍ࠼㸪GSH ࡢࡼ࠺࡞ෆᅉᛶ≀㉁ࡢኚືࢆ
ホ౯ྍ⬟࡞ࣔࢹ࡛ࣝ࠶ࡿ࡜⪃࠼ࡽࢀࡓࠋࡇࡢࡇ࡜࠿ࡽ㸪PXB ࣐࢘ࢫࡣ཯ᛂᛶ௦ㅰ≀࡟ࡼࡿ⫢ẘᛶࡢ࣓࢝ࢽࢬ࣒ゎ᫂ࡸࣜࢫࢡホ౯ࢆ⾜࠺ୖ࡛᭷⏝࡞ࣄࢺ໬ື≀ࣔࢹࣝ࡟࡞ࡿࡶࡢ࡜⪃࠼ࡽࢀࡓࠋᮏ◊✲࡛ᚓࡽࢀࡓ⤖ᯝࡣ㸪FBM ࡢ཯ᛂᛶ௦ㅰ≀⏕ᡂ࣓࢝ࢽࢬ࣒ࡢゎ᫂ཬࡧ⮫ᗋ࡟࠾࠸࡚㸪FBM ࢆࡼࡾᏳ඲࡟౑⏝ࡍࡿୖ࡛᭷⏝࡞ᇶ♏ⓗ▱ぢ࡟࡞ࡿࡶࡢ࡜⪃࠼ࡽࢀࡿࠋ

44

Fig. 16. Postulated mechanisms of reactive metabolite formation and hepatotoxicity by FBM.

45

ㄽᩥ┠㘓

ᮏ༤ኈㄽᩥࡢᇶ♏࡜࡞ࡿཎⴭㄽᩥࢆ௨ୗ࡟♧ࡍࠋ

Sato, K., Sanoh, S., Ishida, Y., Tateno, C., Ohta, S. and Kotake, Y.: Assessment of metabolic activationof felbamate in chimeric mice with humanized liver in combination with in vitro metabolic assays. J.
Toxicol. Sci., 2022, in press.

46

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ᮏ◊✲ࢆ⾜࠺࡟࠶ࡓࡾ㸪⤊ጞࡈ᠓⠜࡞ࡿࡈᣦᑟ㸪ࡈ㠴᧡ࢆ㈷ࡾࡲࡋࡓᗈᓥ኱Ꮫ኱Ꮫ㝔་⣔⛉Ꮫ◊✲⛉ ⏕యᶵ⬟ศᏊືែᏛ◊✲ᐊ ྂṊ ᘺ୍㑻 ᩍᤵࢆࡣࡌࡵ㸪࿴ḷᒣ┴❧་⛉኱Ꮫ⸆Ꮫ㒊 ⾨⏕⸆Ꮫ◊✲ᐊ బ⬟ ṇ๛ ෸ᩍᤵ㸪ኴ⏣ ⱱ ᩍᤵ࡟῝⏒ࡓࡿㅰពࢆ
⾲ࡋࡲࡍࠋᮏ◊✲ࡢ㐙⾜࡟㝿ࡋ㸪᱁ูࡢࡈ㧗㓄ࢆ㈷ࡾࡲࡋࡓᗈᓥ኱Ꮫ኱Ꮫ㝔་⣔⛉Ꮫ◊✲⛉ ᚤ⏕≀⸆ရ㛤ⓎᏛ◊✲ᐊ 㯮⏣ ↷ኵ ᩍᤵ㸪⏕⸆Ꮫ◊✲ᐊ ᒣ㔝 ᖾᏊ ෸ᩍᤵ࡟ཌࡃᚚ♩⏦ࡋୖࡆࡲࡍࠋ
ᮏㄽᩥࡢసᡂ࡟࠶ࡓࡾ㸪ᩘࠎࡢࡈຓゝ㸪ࡈᣦᑟࢆ㈷ࡾࡲࡋࡓᗈᓥ኱Ꮫ኱Ꮫ㝔་⣔⛉Ꮫ◊✲⛉ ་⒪⸆๣Ꮫ◊✲ᐊ 㧗㔝 ᖿஂ ᩍᤵ㸪⸆ຠゎᯒ⛉Ꮫ◊✲ᐊ ᳃ᒸ ᚨග ᩍᤵ㸪᰾㓟ศᯒ໬Ꮫ◊✲ᐊ Ἑྜ ⚽ᙪ ෸ᩍᤵ࡟῝ࡃឤㅰ⮴ࡋࡲࡍࠋࡲࡓ㸪ᮏ◊✲ࡢ㐙⾜࡟ከ኱࡞ࡿࡈᨭ᥼㸪ࡈ༠ຊࢆ㡬ࡁࡲࡋࡓᰴᘧ఍♫ࣇ࢙ࢽࢵࢡࢫࣂ࢖࢜ ❧㔝 ▱ୡ ༤ኈ㸪▼⏣ 㞝஧ ༤ኈ࡟῝ࡃᚚ♩⏦ࡋୖࡆࡲࡍࠋ
ᮏ◊✲ࢆ㐍ࡵࡿ࡟࠶ࡓࡾ㸪ᩘከࡃࡢࡈຓゝࢆ࠸ࡓࡔࡁࡲࡋࡓ࢔ࢫࢸࣛࢫ〇⸆ᰴᘧ఍♫㛗ᆏ Ὀஂ ༤ኈ㸪⚄ᒣ ె㍤ ༤ኈࢆࡣࡌࡵ㸪ᰴᘧ఍♫࣊ࣜ࢜ࢫ ᡂ෠ ὒ୍ ༤ኈ㸪༓ᑑ〇⸆ᰴᘧ఍♫ Ἑᮧ ❶⏕ ༤ኈ࡟ᚚ♩⏦ࡋୖࡆࡲࡍࠋ

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㸸≉␗య㉁ᛶ⸆≀ẘᛶ. ᪥⸆⌮ㄅ㸪127㸪473480.

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