ROS ࢔ࢵࢭ࢖ホ౯ሗ࿌᭩2015 ᖺ 7 ᭶ 8 ᪥

JaCVAM ගẘᛶ㈨ᩱ⦅⧩ጤဨ఍7ጤဨ㛗ᇼ஭ 㑳ኵ

㸦ࣇ࢓࢖ࢨ࣮ᰴᘧ఍♫㸧ጤ ဨᑿୖ ㄔⰋ

㸦㟼ᒸ┴❧኱Ꮫ㸧㔠Ꮚ ࿴ᘯ

㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧ୖ᭶ ⿱୍

㸦ᰴᘧ఍♫ ㈨⏕ᇽ ⏣୰ ᠇✑㸦㣗ရ⸆ရᏳ඲ࢭࣥࢱ࣮㸧➜ᮌ ಟ

㸦་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧⣽஭ ୍ᘯ

㸦ཧኳ〇⸆ᰴᘧ఍♫㸧

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┠ḟ
i.
␎ㄒ ...................................................................................................................... 4ii.
せ᪨ ...................................................................................................................... 5
1.
ヨ㦂᪉ἲࡢ⛉Ꮫⓗ⫼ᬒ࣭᰿ᣐ࠾ࡼࡧつไୖࡢ఩⨨࡙ࡅ ....................................... 6
1.1⛉Ꮫⓗ⫼ᬒ............................................................................................................ 6
1.2⛉Ꮫⓗ᰿ᣐ............................................................................................................ 7
1.3
つไୖࡢ఩⨨࡙ࡅ ................................................................................................. 92ヨ㦂ࡢࣉࣟࢺࢥࣝ࠾ࡼࡧุᐃᇶ‽ ..................................................................... 11
2.1
ᐃཎ⌮ ............................................................................................................. 11
2.2ᮦᩱ࠾ࡼࡧヨ⸆ .................................................................................................. 11
2.3⿕㦂≀㉁ ............................................................................................................. 12
2.4
㝧ᛶ࡞ࡽࡧ࡟㝜ᛶᑐ↷≀㉁ ................................................................................ 13
2.5
ჾල࣭ᶵჾ.......................................................................................................... 13
2.6
ヨ㦂᪉ἲ࡞ࡽࡧ࡟ࢹ࣮ࢱゎᯒ............................................................................. 14
2.7
ࢹ࣮ࢱ᥇⏝᮲௳ .................................................................................................. 17
2.8
㝧ᛶᇶ‽ ............................................................................................................. 183
ヨ㦂ἲࡢࣂࣜࢹ࣮ࢩࣙࣥ .................................................................................... 18
3.1⏝࠸ࡽࢀࡓ≀㉁࡜ࡑࡢጇᙜᛶ............................................................................. 18
3.2ࣂࣜࢹ࣮ࢩࣙࣥ⤖ᯝࡢホ౯ ................................................................................ 214
ヨ㦂᪉ἲ࡟㛵ࡍࡿ௚ࡢ⛉Ꮫⓗ⪃ᐹ ..................................................................... 25
4.1ග࡟୙Ᏻᐃ࡞≀㉁࡬ࡢ㐺⏝ ................................................................................ 25
4.2
㞴⁐ᛶ≀㉁࡬ࡢᑐᛂ ........................................................................................... 25
4.3
ΰྜ≀࠾ࡼࡧศᏊ㔞୙᫂ヨᩱ࡬ࡢᑐᛂ .............................................................. 26
4.4ග࢔ࣞࣝࢠ࣮ᛶண ࡬ࡢ฼⏝............................................................................. 28
5
3Rs ࡬ࡢ㛵୚㸦ື≀⚟♴㠃࠿ࡽࡢጇᙜᛶ㸧 ....................................................... 296ヨ㦂᪉ἲࡢ᭷⏝ᛶ࡜ၥ㢟Ⅼ ................................................................................ 29
7⤖ㄽ .................................................................................................................... 308⏝ㄒࡢゎㄝ.......................................................................................................... 319ཧ⪃ᩥ⊩ ............................................................................................................. 31
10
Appendix............................................................................................................. 34
Appendix 1 Executive Summary of “Peer Review Panel Evaluation of the Reactive Oxygen
Species (ROS) Photosafety Assay” (16 October 2013) ..................................... 34
Appendix 2 List of reasons for chemical selection .................................................................. 36
Appendix 3 Test chemicals for the Phase 1 study .................................................................. 38
Appendix 4 List of reasons for chemical selection .................................................................. 39
Appendix 5 Test chemicals for Phase 2 study and code list................................................... 45





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i.

␎ㄒ
3T3 NRU-PT:

3T3 neutral red uptake phototoxicity test

CDER:
CPMP:

Center for Drug Evaluation and Research㸦་⸆ရホ౯◊✲ࢭࣥࢱ࣮㸧
Committee for Proprietary Medicinal Products㸦Ḣᕞ་⸆ရጤဨ఍㸧

DMSO:
EMEA:

Dimethyl sulfoxide
European Agency for the Evaluation of Medicinal Products㸦Ḣᕞ་⸆ရᑂᰝᗇ㸪2004 ᖺࡲ࡛౑ࢃࢀ࡚࠸ࡓ࿧⛠㸧

EMA:
EWG:

European Medicines Agency㸦Ḣᕞ་⸆ရᗇ㸧
Expert Working Group㸦ᑓ㛛ᐙసᴗ㒊఍㸧

FDA

Food and Drug Administration㸦⡿ᅜ㣗ရ་⸆ရᒁ㸧

IC:
ICH:

Internal conversion㸦ෆ㒊㌿⛣㸧International Conference on Harmonization of Technical

ISC:

Requirements for Registration of Pharmaceuticals for Human Use㸦᪥⡿ EU ་⸆ရつไㄪ࿴ᅜ㝿఍㆟㸧
Intersystem crossing㸦㡯㛫஺ᕪ㸧

NaPB:
NBT:
NBT+:
OECD:
RNO:
ROS:
UVA:
UVB:
UVC:
UV/VIS:

Sodium Phosphate Buffer
Nitroblue tetrazolium
Monoformazan
Organisation for Economic Co-operation and Development㸦⤒῭༠ຊ㛤Ⓨᶵᵓ㸧p-nitrosodimethyl aniline
Reactive Oxygen Species, including superoxide anion (SA) andsinglet oxygen (SO).
Ultraviolet light A (wavelengths between 320 and 400 nm).
Ultraviolet light B (wavelengths between 290 and 320 nm).
Ultraviolet light C (wavelengths between 190 and 290 nm).
Ultraviolet and visible light

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LL

せ᪨
Reactive oxygen species (ROS) ࢔ࢵࢭ࢖ࡣ㸪໬Ꮫ≀㉁ࡢග໬Ꮫⓗ≉ᛶࢆᣦᶆ࡜
ࡋࡓ⡆౽࡛ከ᳨యฎ⌮ྍ⬟࡞ගᏳ඲ᛶホ౯ἲ࡜ࡋ࡚ᥦ᱌ࡉࢀ㸪໬Ꮫ≀㉁࡟ᨃఝ
ኴ㝧ගࢆ↷ᑕࡋ㸪singlet oxygen ࡜ super oxide anion ࡢ⏘⏕ࡢ᭷↓ࢆㄪ࡭ࡿග཯
ᛂᛶヨ㦂ࡢ୍ࡘ࡜ࡋ࡚㸪ග⥺㐣ᩄ⑕ㄏⓎࡢྍ⬟ᛶࢆண ࡋᚓࡿࡇ࡜ࡀ᫂ࡽ࠿࡟
ࡉࢀ࡚࠸ࡿ㸬ࡇࡢ ROS ࢔ࢵࢭ࢖ࡣ 2014 ᖺ࡟ step 5 ࡟฿㐩ࡋࡓ ICH S10 ࢞࢖ࢻࣛ࢖ࣥ࡟࠾࠸࡚ࡶ࡜ࡾୖࡆࡽࢀ࡚࠾ࡾ
ࠕࢹ࣮ࢱ࠿ࡽࡣ㸪ࡇࡢヨ㦂ἲࡣ in vivo ࡟࠾ࡅࡿ┤᥋ⓗ࡞ගẘᛶ≀㉁ࢆணぢࡍࡿୖ࡛ࡢឤᗘࡀ㧗࠸ࡇ࡜ࡀ♧ࡉࢀ࡚࠸ࡿࠖ
࡜グ㏙ࡉࢀ࡚࠸ࡿ㸬
ROS ࢔ࢵࢭ࢖ࡢ᭷⏝ᛶホ౯࡜ࡋ࡚㸪JaCVAM ᣦᑟୗ࡛ࡢከ᪋タࣂࣜࢹ࣮ࢩࣙ
ࣥヨ㦂ࡀᐇ᪋ࡉࢀࡓ⤖ᯝ㸪᪋タෆ࠾ࡼࡧ᪋タ㛫ᕪࡀᑡ࡞ࡃ㸪ỗ⏝ᛶ࡟ඃࢀ࡚࠸
ࡿヨ㦂ἲ࡛࠶ࡿࡇ࡜ࡀ♧ࡉࢀࡓ㸬ࡲࡓ㸪㞴⁐ᛶࡢࡓࡵホ౯࡛ࡁ࡞࠿ࡗࡓ⿕㦂≀㉁ࢆ㝖ࡃ࡜㸪ROS ࢔ࢵࢭ࢖࡟ࡼࡿගẘᛶ≀㉁ࡢ㝧ᛶ᳨ฟ⋡ࡣ 100%࡛࠶ࡾ㸪ഇ㝜ᛶ⤖ᯝࡀ࡞࠸ࡇ࡜࠿ࡽ㸪ROS ࢔ࢵࢭ࢖ࡣගẘᛶ࣏ࢸࣥࢩࣕࣝࡢホ౯࡟᭷⏝࡛࠶ࡿ࡜⪃࠼ࡽࢀࡓ㸬ࡇࢀࡽࡢࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩࡟ᑐࡍࡿ➨୕⪅ホ౯࡟࠾࠸࡚
ࡶྠᵝࡢホ౯ࡀ࡞ࡉࢀࡓ㸬ࡇࡢࡼ࠺࡞⫼ᬒࡢࡶ࡜㸪JaCVAM ࡣ OECD ࡀᐃࡵࡓᅜ㝿ⓗ࡞ᇶ‽㸦2005 ᖺ㸧࡟ᚑࡗ࡚㸪ROS ࢔ࢵࢭ࢖ࡢࣂࣜࢹ࣮ࢩࣙࣥ≧ἣ࡟㛵ࡋ࡚⛉Ꮫⓗ࡞ホ౯ࡸ᳨ドࢆ⾜࠺ࡓࡵࡢᑓ㛛ᐙ࡟ࡼࡿ⊂❧ࡋࡓጤဨ఍ࢆタࡅࡓ㸬ጤဨ఍ࡣ 2013 ᖺ 2 ᭶࡟➨ 1ᅇࡢ఍ྜࢆᣢࡕ㸪ࡑࡢᚋ 5 ḟ࡟ரࡗ࡚ホ౯ሗ࿌᭩ࡢぢ┤ࡋࢆ㔜ࡡ࡚ࡁࡓ㸬ᮏホ౯ሗ࿌᭩ࡣጤဨ఍࡟ࡼࡿホ౯࡜⤖ㄽࢆせ⣙ࡋࡓࡶࡢ࡛࠶ࡿ㸬ᙜጤဨ఍ࡣ⤖ㄽ࡜ࡋ࡚㸪ROS ࢔ࢵࢭ࢖ࡢ෌⌧ᛶ࡜ண ᛶࡣ໬Ꮫ≀㉁ࡢ⥲ྜⓗ࡞ගẘᛶホ౯ୖ㸪༑ศ࡟㐺ᛂ࡛ࡁࡿ࢔ࢵࢭ࢖᪉ἲ࡛࠶ࡿ࡜⤖ㄽࡋࡓ㸬௒ᚋ㸪ROS࢔ࢵࢭ࢖࡟࠾࠸࡚㝧ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜ㸪ḟࡢẁ㝵࡜ࡋ࡚ 3T3 ගẘᛶヨ㦂㸦OECD ヨ㦂࢞࢖ࢻࣛ࢖ࣥ#432㸧➼ࡢ in vitro ヨ㦂ࡢ㐺⏝ࡀ᥎ዡࡉࢀࡿࡀ㸪㝜ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜࡣ᭦࡞ࡿື≀ヨ㦂ࡸࡑࡢ௚ࡢヨ㦂ࡣ୙せ࡛࠶ࡿ࡜ᖐ⤖࡛ࡁࡿࡇ࡜࡟ࡼࡾ㸪ගẘᛶホ౯࡟ᚲせ࡜࡞ࡿ᫬㛫ࡸ㈝⏝ࢆ㍍ῶࡋ㸪ᐇ㦂ື≀ࡢ౑⏝ࢆ๐ῶࡍࡿࡇ࡜ࡀ࡛ࡁࡿ࡜⪃࠼ࡽࢀࡿ㸬ࡲࡓ㸪ᮏ࢔ࢵࢭ࢖ࡢண ᛶ࣭෌⌧ᛶࡣ㸪་⸆ရࡢගẘᛶヨ㦂ἲࡢ୍⎔࡜ࡋ࡚㸪ࡑࡢ๰⸆ึᮇẁ㝵࠾ࡼࡧ㛤ⓎࡢពᛮỴᐃ࡟⏝࠸ࡿ஦ࢆᨭᣢ࡛ࡁࡿࡶࡢ࡛࠶ࡿ㸬ࡉࡽ࡟㸪᭱㏆ࡢ᳨ウ࡟࠾࠸࡚㸪ROS ࢔ࢵࢭ࢖࡟࠾ࡅࡿΰྜ≀࠾ࡼࡧศᏊ㔞୙᫂
ヨᩱ࡬ࡢ㐺⏝ྍ⬟ᛶࡀ♧ࡉࢀ㸪໬⢝ရཎᩱ㸪୍⯡໬Ꮫ≀㉁࡞ࡽࡧ࡟㎰⸆ࡢගᏳ඲ᛶ࡟㛵ࡍࡿࣁࢨ࣮ࢻ᳨ฟἲ࡜ࡋ࡚ ROS ࢔ࢵࢭ࢖࡬ࡢᮇᚅࡀ㧗ࡲࡗ࡚࠸ࡿ㸬511

 ヨ㦂᪉ἲࡢ⛉Ꮫⓗ⫼ᬒ࣭᰿ᣐ࠾ࡼࡧつไୖࡢ఩⨨࡙ࡅ
1.1

⛉Ꮫⓗ⫼ᬒ

 ගᏳ඲ᛶ࡜ࡣ㸪ගࢆ௓ࡋ࡚Ⓨ⑕ࡍࡿ໬Ꮫ≀㉁ㄏⓎᛶࡢẘᛶ཯ᛂ࡛࠶ࡾ㸪ᅗ 1(A)࡟♧ࡋࡓࡼ࠺࡟ග࡟ࡼࡗ࡚໬Ꮫ≀㉁ࡀບ㉳ࡍࡿࡇ࡜ࡀࢺ࣮ࣜ࢞࡜࡞ࡗ࡚࠸ࡿ㸬ࡇࡢࡼ࠺࡞ẘᛶࢆ♧ࡍ໬ྜ≀ࢆᅗ 1(B)࡛ࡣບ㉳ගẘᛶ໬ྜ≀࡜ᐃ⩏ࡋ࡚࠸ࡿࡀ㸪ࡇࡢࡼ࠺࡞≀㉁ࡣ㸪ග่⃭ᛶ㸪ග࢔ࣞࣝࢠ࣮ᛶ㸪ග㑇ఏẘᛶ㸪ගࡀࢇཎᛶࢆㄏᑟࡍࡿྍ⬟ᛶࡀ࠶ࡿ㸬໬Ꮫ≀㉁ࡢගᏳ඲ᛶࢆホ౯ࡍࡿࡓࡵ࡟ᵝࠎ࡞࢔ࢵࢭ࢖ἲ㸦in vitro ࠾ࡼࡧ in vivo㸧ࡀ㛤Ⓨࡉࢀ࡚࠾ࡾ㸪ࡇࢀࡽ࡟ࡣ㸪(i) in silico ࢫࢡ࣮ࣜࢽࣥࢢࢶ࣮ࣝ㸪(ii) ග໬Ꮫⓗ≉ᛶࢆ୰ᚰ࡜ࡋࡓศᏊ≀ᛶホ౯㸪(iii) ྛ✀ẘᛶ཯ᛂ࡟≉␗ⓗ࡞ග⏕≀໬Ꮫⓗ࢔ࢵࢭ࢖᪉ἲ࡞࡝ࡀྵࡲࢀ࡚࠸ࡿ (Onoue et al., 2009)㸬
in silico ホ౯ࢩࢫࢸ࣒࡜ࡋ࡚ࡣ㸪౛࠼ࡤ DEREK ࡸ HOMO-LUMO Gap ➼ࡀ▱ࡽࢀ࡚࠸ࡿ㸬ග໬Ꮫⓗ≉ᛶホ౯ࢶ࣮ࣝࡣᐇ㝿࡟ྜᡂࡉࢀࡓ་⸆ရೃ⿵໬ྜ≀ࡢศᏊ≀ᛶࢆᣦᶆ࡜ࡋࡓග
ឤཷᛶศᯒࢆ୺య࡜ࡋ㸪⸆๣ᛶග⥺㐣ᩄ⑕ࡢㄏⓎ࡟ᐤ୚ࡋᚓࡿග໬Ꮫⓗ཯ᛂࡢ᭷↓࡟ࡘ࠸࡚㧗࠸ࢫ࣮ࣝࣉࢵࢺ࡛♧၀ࡍࡿࡇ࡜ࡀฟ᮶ࡿ㸬ගẘᛶࡢᶵᗎࢆ⪃࠼ࡿ࡜ࡁ㸪᭱ࡶ㔜せ࡞ࢺ࣮ࣜ࢞࡜࡞ࡿࡢࡣኴ㝧ගࡢ྾཰㸪ࡑࡋ࡚ࡑࢀ࡟క࠺໬ྜ≀ࡢບ㉳࡛࠶ࢁ࠺㸬ࡇࡢほⅬ࠿ࡽ㸪ࠕኴ㝧ගࡢ྾཰ࡋࡸࡍࡉࠖࡢᣦᶆ࡛࠶ࡿ UV/VIS ྾཰≉ᛶࡀ་⸆ရ㛤Ⓨ࡟࠾ࡅࡿගᏳ඲ᛶホ౯࡟࠾࠸࡚᭷⏝࡛࠶ࡿࡇ࡜ࡀ Henry ࡽ࡟ࡼࡗ࡚ᥦ᱌ࡉࢀࡓ㸬Henry ࡽࡢሗ࿌࡟ࡼࢀࡤ㸪ࣔࣝ྾ගಀᩘ 1,000 M-1㺃cm-1 ௨ୗࡢ໬ྜ≀ࡣගẘᛶࣜࢫࢡࡀప࠸ (Henry et al., 2009)㸬ࡉࡽ࡟㸪Bauer ࡽࡣࡇࡢ㜈್࡟ࡘ࠸࡚ࡼࡾከࡃࡢ໬Ꮫ≀㉁ࢆᑐ㇟࡟⢭ᰝࡋ㸪ࡑࡢጇᙜᛶ࡟ࡘ࠸᳨࡚ドࡋ࡚࠸ࡿ (Bauer et al., 2014)㸬୍᪉㸪UV/VIS ྾཰≉ᛶࡔࡅ࡛࡞ࡃ㸪ບ㉳࢚ࢿࣝࢠ࣮࡟
ࡼࡿග໬Ꮫⓗ཯ᛂᛶࢆ┤᥋ホ౯ࡍࡿࡇ࡜ࡀྍ⬟࡜࡞ࢀࡤ㸪ࡼࡾᐇ㉁ⓗ࡞ගẘᛶண࡟ᐤ୚࡛ࡁࡿྍ⬟ᛶࡀ࠶ࡿ㸬ࡑࡇ࡛㸪Onoue ࡽࡣࡇࡢほⅬ࠿ࡽග↷ᑕୗ࡟࠾ࡅࡿ⸆≀࠿ࡽࡢ ROS⏘⏕ࢆᣦᶆ࡜ࡋࡓ᪂ࡓ࡞ගẘᛶணࢶ࣮࡛ࣝ࠶ࡿ ROS ࢔ࢵࢭ࢖ࢆ 2006 ᖺ࡟⪃᱌ࡋࡓ(Onoue and Tsuda, 2006)㸬ࡍ࡛࡟ୖᕷࡉࢀ࡚࠸ࡿ」ᩘࡢ་⸆ရࢆࡑࢀࡒࢀ ROS ࢔ࢵࢭ࢖࡛ホ౯ࡋࡓ࡜ࡇࢁ㸪๪స⏝ሗ࿌࡜ࡋ࡚ග⥺㐣ᩄ⑕ࡀሗ࿌ࡉࢀ࡚࠸ࡿ໬ྜ≀⩌ࡣ ROS ࢆ⏘⏕ࡍࡿഴྥ࡟࠶ࡾ㸪୍᪉㸪ᵝࠎ࡞ࢫࢡ࣮ࣜࢽࣥࢢ࡛ගᏳ඲ᛶࡢ㧗࠸࡜⪃࠼ࡽࢀࡿ໬ྜ≀⩌࠿ࡽࡢ ROS ⏘⏕ࡣᴟࡵ࡚㝈ᐃࡉࢀࡓࡶࡢ࡛࠶ࡗࡓ㸬ࡍ࡞ࢃࡕ㸪ROS ࢔ࢵࢭ࢖ࡣගẘᛶࣜࢫࢡࢆணࡍࡿ࠺࠼࡛᭷⏝࡞ࢶ࣮ࣝ࡜࡞ࡿྍ⬟ᛶࡀ♧၀ࡉࢀ㸪ᮏ࢔ࢵࢭ࢖ࡢࣁ࢖ࢫ࣮ࣝࣉࢵࢺ

ࢫࢡ࣮ࣜࢽࣥࢢ⣔࡬ࡢᛂ⏝᳨ウࡢ⤖ᯝ㸪ࢫ࣮ࣝࣉࢵࢺࡢᨵၿ࡜࢔ࢵࢭ࢖ࡢᑠࢫࢣ࣮ࣝ໬࡟
ᡂຌࡋ࡚࠸ࡿ (Onoue et al., 2008a)㸬ROS ࢔ࢵࢭ࢖ࡢ฼Ⅼ࡜ࡋ࡚ࡣ㸪໬Ꮫ≀㉁ࡢග໬Ꮫⓗ཯ᛂᛶࢆ㎿㏿࠿ࡘ⡆౽࡟ᐃ࡛ࡁࡿࡇ࡜ࡀᣲࡆࡽࢀࡿࡀ㸪ࡑࡢ཯㠃㸪ᮏ࢔ࢵࢭ࢖ࢹ࣮ࢱࡣග

Ᏻ඲ᛶࡔࡅ࡛ࡣ࡞ࡃ㸪ගᏳᐃᛶ࡞࡝࡟ၥ㢟ࡀ࠶ࡿ໬ྜ≀ࡶྵࡵ࡚㝧ᛶ࡜ุูࡋ࡚ࡋࡲ࠺ࡇ
࡜ࡀㄢ㢟ࡢ୍ࡘ࡛࠶ࡾ㸪ࡇࢀࡣග໬Ꮫⓗ཯ᛂᛶホ౯ἲ࡛࠶ࡿࡀᨾࡢㄢ㢟࡛࠶ࡿ㸬612

 ᮏᩥ᭩࡟࠾࠸࡚㸪ගᏳ඲ᛶ࡜ࡣ໬Ꮫ≀㉁࡟᥋ゐ࠶ࡿ࠸ࡣᦤྲྀࡋࡓᚋ࡛㸪ග࡬ࡢ᭚㟢࡟㛵㐃ࡋ࡚Ⓨ⑕ࡍࡿ᭷ᐖస⏝඲⯡ࢆᑐ㇟࡜ࡋ㸪ᚑ᮶ࡼࡾ㸪ගẘᛶ㸪ග࢔ࣞࣝࢠ࣮ᛶ㸪ග㑇ఏẘᛶ㸪ගࡀࢇཎᛶࡢ 4 ✀ࡢ␗࡞ࡿస⏝ࡀྵࡲࢀࡿ㸬ගᏳ඲ᛶࡣ㸪ᑐ㇟࡜࡞ࡿ໬Ꮫ≀㉁ࡢග໬
Ꮫⓗ≉ᛶ㸪㠀⮫ᗋヨ㦂ࡢࢹ࣮ࢱ࠾ࡼࡧ⮫ᗋᏳ඲ᛶ᝟ሗࢆࡩࡲ࠼࡚⤫ྜⓗ࡟ホ౯ࡉࢀࡿ㸬ගẘᛶࡣග่⃭ᛶ࡜ྠ⩏࡛㸪ග↷ᑕ࡟ࡼࡗ࡚⏘⏕ࡉࢀࡿග཯ᛂᛶ≀㉁࡟ᑐࡍࡿᛴᛶࡢ⤌⧊཯ᛂ࡛࠶ࡿ㸬ග⥺㐣ᩄ⑕࡜ࡣ㸪ග↷ᑕ࡟ࡼࡾច㉳ࡉࢀࡿ⤌⧊཯ᛂ࡟ᑐࡋ㸪᫬ᢡ౑⏝ࡉࢀࡿ୍⯡⏝ㄒ࡛࠶ࡾ㸪ᗈ⩏ࡢගẘᛶ࡜ྠࡌព࿡࡛⏝࠸ࡽࢀࡿࡇ࡜ࡶ࠶ࡿࡀ㸪ගẘᛶ࡜ග࢔ࣞࣝࢠ
࣮ᛶࢆ᫂☜࡟༊ูࡍࡿᚲせࡀ࠶ࡿሙྜ࡟౑⏝ࡍࡿࡇ࡜ࡣ㐺ษ࡛ࡣ࡞࠸㸬
ROS ࢔ࢵࢭ࢖ࣂࣜࢹ࣮ࢩ࣐ࣙࣥࢿࢪ࣓ࣥࢺࢳ࣮࣒ࡣ JaCVAM ᣦᑟୗ࡛」ᩘࡢཧຍ᪋タ࡛ 2 ✀㢮ࡢᨃఝኴ㝧ග↷ᑕ⿦⨨ࢆ⏝࠸ࡓࣂࣜࢹ࣮ࢩࣙࣥヨ㦂 2 ヨ㦂ࢆᐇ᪋ࡋ㸪ROS ࢔ࢵࢭ࢖ࡢᢏ⾡⛣㌿ᛶ㸦transferability㸧㸪ሀ∼ᛶ㸦robustness㸧࡞ࡽࡧ࡟ண

ᛶ㸦prediction capacity㸧ࢆ᳨ドࡋࡓ㸬ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂⤖ᯝࢆㄽᩥ࡜ࡋ࡚ᢞ✏ࡋ(Onoue et al., 2013a; Onoue et al.,2014a)㸪ヲ⣽࡞ࢹ࣮ࢱࢆྵࡴࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩࡞ࡽࡧ࡟ヨ㦂ἲࣉࣟࢺࢥ࣮ࣝࢆ JaCVAM
࣮࣒࣮࣍࣌ࢪୖ࡟බ㛤ࡋࡓ㸦ROS assay validation management team, 2013a, 2013b, 2013c㸧㸬
ROS ࢔ࢵࢭ࢖ࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩ࢆホ౯ࡋࡓ➨୕⪅ホ౯ጤဨ఍ࡢሗ࿌᭩ࡶ JaCVAM ࣮࣒࣮࣍࣌ࢪୖ࡟බ㛤ࡉࢀ㸦Spielmann et al., 2013㸧㸪ࡑࡢせ⣙ࢆ Appendix 1 ࡟♧ࡋࡓ(Appendix
1: Executive Summary of ͆Peer Review Panel Evaluation of the Reactive Oxygen Species (ROS)
Photosafety Assay”).

1.2

⛉Ꮫⓗ᰿ᣐ

 ࡝ࡢࡼ࠺࡞Ⓨ⑕ᶵᗎࡢගẘᛶ཯ᛂ࡛ࡶ㸪ࡑࡢⓎ⑕ᶵᗎࡢୖὶ࡟ࡣග໬Ꮫ཯ᛂࡀᏑᅾࡍࡿ㸬
1817 ᖺ࡟ᥦၐࡉࢀࡓග໬Ꮫ➨୍ἲ๎ (ࡲࡓࡣ Grotthuss-Draper law) ࡛ࡣ ͆ධᑕࡋࡓගࡢ࠺
ࡕ㸪྾཰ࡉࢀࡓࡶࡢࡔࡅࡀ཯ᛂ࡟㛵ࢃࡿ͇ ࡜࠶ࡾ㸪ࡍ࡞ࢃࡕග྾཰ࡍࡿ໬ྜ≀ࡢࡳࡀගẘᛶ཯ᛂࢆ♧ࡋᚓࡿࡇ࡜ࡀ㏙࡭ࡽࢀ࡚࠸ࡿ㸬࢜ࢰࣥᒙࢆ㏻ࡗ࡚ᆅ⾲࡟฿㐩࡛ࡁࡿኴ㝧ගࡢἼ㛗ࡣ୺࡟ 290̽700 nm ⛬ᗘ࡛࠶ࡿࡀ㸪300 nm ࡼࡾࡶ▷Ἴ㛗࡛ẚ㍑ⓗ࢚ࢿࣝࢠ࣮ࡢ㧗࠸ගࡢ┿⓶฿㐩⋡ࡣࢃࡎ࠿ 10% ⛬ᗘ࡛࠶ࡿ㸬୍᪉㸪㛗Ἴ㛗ഃ࡟࡞ࡿ࡟ࡘࢀ࡚࢚ࢿࣝࢠ࣮ࡣᑠࡉࡃ
࡞ࡿࡶࡢࡢ㸪⓶⭵㏱㐣ᛶࡣ㧗ࡃ࡞ࡾ㸪ࡇࢀࡽࡀ⸆๣ᛶග⥺㐣ᩄ⑕ࡢࢺ࣮ࣜ࢞࡜࡞ࡿࡇ࡜ࡶ
ከ࠸㸬యෆ࡟ྲྀࡾ㎸ࡲࢀࡓගẘᛶ≀㉁ࡣࡲࡎ⓶⭵⤌⧊࡟฿㐩ࡋ㸪⸆๣ࡢศᏊෆ chromophore㸪࠶ࡿ࠸ࡣ௦ㅰ࡟ࡼࡗ࡚⋓ᚓࡉࢀࡓ chromophore ࡀ⓶⭵῝㒊ࡲ࡛฿㐩ࡋࡓග࡟ࡼࡗ࡚↷ᑕࡉࢀࡿ࡜㸪ᇶᗏ≧ែࡢ S0 ࠿ࡽບ㉳୍㔜㡯≧ែ S1 ࡟ບ㉳ࡉࢀࡿ㸦ᅗ 1(A)㸧㸬ບ㉳୍㔜㡯≧ែࡢᑑ࿨ࡣ▷ࡃ㸪ࡍ࡞ࢃࡕ⺯ගࢆⓎࡋ࡚┤ࡕ࡟ᇶᗏ≧ែ S0 ࡟ᡠࡿ࠿㸪㡯㛫஺ᕪ࡟ࡼࡾບ
㉳୕㔜㡯≧ែ T1 ࡟㑄⛣ࡍࡿ㸬ບ㉳୕㔜㡯≧ែ࡟࠶ࡿ໬ྜ≀ࡣࡾࢇගࢆⓎࡋ࡚ᇶᗏ≧ែ S0࡟ᡠࡿ㸬ࡇࡢࡼ࠺࡟ගࢆ྾཰ࡋ࡚ບ㉳ࡉࢀࡓศᏊࡣ㏿ࡸ࠿࡟ࡶ࡜ࡢᇶᗏ≧ែ࡟ᡠࡿ≉ᛶࡀ࠶ࡿࡀ㸪ࡇࡢ㐣⛬ࡀᴟࡵ࡚ࢫ࣒࣮ࢬ࡟㉳ࡇࢀࡤ྾཰ࡉࢀࡓග࢚ࢿࣝࢠ࣮࡟ࡼࡿගẘᛶ཯ᛂ࡬ࡢᐤ୚ࡣ㝈ᐃࡉࢀࡓࡶࡢ࡜࡞ࡿ㸬ࡋ࠿ࡋ㸪ᇶᗏ≧ែ࡛ࡣࡲࡗࡓࡃ໬Ꮫ཯ᛂࢆࡋ࡞࠸᮲௳

713࡛ࡶ㸪㧗࠸ග࢚ࢿࣝࢠ࣮ࢆ⋓ᚓࡋࡓບ㉳ศᏊࡣ㸪ࡑࡢ࢚ࢿࣝࢠ࣮ࢆ㥑ືຊ࡜ࡋ࡚⤖ྜࡢゎ⿣ࡸ⏕ᡂࡲࡓࡣ⤌ࡳ᥮࠼࡞࡝ࡢ໬Ꮫ཯ᛂࢆ㉳ࡇࡍࡇ࡜ࡀ࡛ࡁࡿ㸬ࡑࡢࡼ࠺࡞㐣⛬ࢆග໬Ꮫ㐣⛬࡜࠸࠸㸪ࣛࢪ࢝ࣝ཯ᛂ࡛࠶ࡿ Type I ཯ᛂ࡜㸪singlet oxygen ཯ᛂ࡛࠶ࡿ Type II ཯ᛂ࡜࡟ศࡅࡽࢀࡿ㸬ࡇࢀࡽࡣ⸆๣ᛶග⥺㐣ᩄ⑕ࡢⓎ⑕࡟࠾࠸࡚ึᮇ࡟ㄆࡵࡽࢀ㸪ࡍ࡞ࢃࡕ UV/VISࡢ᭚㟢࡟ࡼࡗ࡚ບ㉳ࡉࢀ㸪ࡑࡢ㝿࡟ᚓࡽࢀࡓບ㉳࢚ࢿࣝࢠ࣮ࡣ⏕యෆ≀㉁ࡸ㓟⣲ศᏊ࡟㌿⛣ࡋࡓࡾ㸪࠶ࡿ࠸ࡣ໬ྜ≀ࡑࡢࡶࡢࡀ⏕యෆ≀㉁࡜⤖ྜࡋ࡚ග௜ຍ≀ࢆᙧᡂࡍࡿ㸬ࡲࡓ㸪㓟⣲ศᏊࡣບ㉳࢚ࢿࣝࢠ࣮ࡢ࢔ࢡࢭࣉࢱ࣮࡜ࡋ࡚ᶵ⬟ࡋ㸪ࡑࢀ࡟క࠸⏘⏕ࡉࢀࡓ singlet
oxygen ࡸ superoxide ➼ࡢάᛶ㓟⣲✀࡟ࡼࡿ⏕యෆ≀㉁ࡢ㓟໬཯ᛂࡀ⸆๣ᛶග⥺㐣ᩄ⑕ࡢ
Ⓨ⑕ཎᅉࡢ୍ࡘ࡜ࡋ࡚⪃࠼ࡽࢀ࡚࠸ࡿ㸦ᅗ 1(B㸧㸧㸬ࡇࢀࡽࡢග໬Ꮫ཯ᛂࡢᶆⓗࡀ⣽⬊⭷ୖࡢྛ✀⏕యᡂศ࡛࠶ࡿሙྜ࡟ࡣග่⃭ᛶࢆㄏⓎࡋ㸪ࡲࡓ DNA ࡢ㓟໬࠶ࡿ࠸ࡣሷᇶಟ㣭࡟ࡼࡗ࡚ග㑇ఏẘᛶࡸගࡀࢇཎᛶࡀⓎ⌧ࡍࡿ㸬ບ㉳ࡉࢀࡓ⸆≀ࡀࣁࣉࢸࣥ࡜࡞ࡾࢱࣥࣃࢡ㉁࡜ග௜ຍ≀ࢆᙧᡂࡋࡓ㝿࡟ࡣ㸪ච␿ཎᛶࢆ♧ࡍࡇ࡜࡟࡞ࡾ㸪᭱⤊ⓗ࡟ග࢔ࣞࣝࢠ࣮ᛶ཯ᛂ
ࢆច㉳ࡍࡿࡶࡢ࡜⪃࠼ࡽࢀࡿ㸬ࡇࡢࡼ࠺࡟㸪⸆≀ࡀບ㉳ࡉࢀࡓᚋ࡟཯ᛂࡍࡿศᏊ✀࡟ࡼࡗ
࡚Ⓨ⌧ࡍࡿẘᛶ཯ᛂࡀࡑࢀࡒࢀ␗࡞ࡿ㸬ࡇࢀࡽࡢᶵᗎ࡟ຍ࠼࡚㸪ບ㉳࢚ࢿࣝࢠ࣮ࡸάᛶ㓟⣲✀࡟ࡼࡗ࡚⸆≀ࡑࡢࡶࡢࡀศゎࡉࢀ㸪ẘᛶ≀㉁ࢆ⏕ᡂࡍࡿࢣ࣮ࢫࡶ᝿ᐃࡉࢀ㸪ẘᛶᶵᗎࡣከᵝ࠿ࡘ」㞧࡛࠶ࡿ㸬ࡇࡢࡼ࠺࡞ග໬Ꮫⓗ཯ᛂࡀගẘᛶ཯ᛂࡢࢺ࣮ࣜ࢞࡟࡞ࡗ࡚࠸ࡿࡇ
࡜ࢆ⪃៖ࡍࢀࡤ㸪ROS ࢔ࢵࢭ࢖ࢆගẘᛶホ౯ࢶ࣮ࣝࡢ୍ࡘ࡜ࡋ࡚฼⏝ࡍࡿࡇ࡜ࡣጇᙜ࡜⪃
࠼ࡽࢀࡿ㸬

ᅗ 1

ගẘᛶᶵᗎ㸦௬ㄝ㸧㸬(A) ග࡟ࡼࡿ໬Ꮫ≀㉁ࡢບ㉳࡜ᇶᗏ≧ែ࡬ࡢ㑄⛣㸬(B) ບ㉳໬ྜ≀ࡢᶆⓗ࡜࡞ࡿ⏕యෆศᏊ࡜ගẘᛶ཯ᛂ㸬(ᑿୖ, 2011)

8
14

1.3

つไୖࡢ఩⨨࡙ࡅ

๓㏙ࡢࡼ࠺࡟㸪ග↷ᑕ࡟ࡼࡗ࡚ບ㉳ගẘᛶ≀㉁ࢆⓎ⏕ࡍࡿ໬ྜ≀ࡣ㸪ග่⃭ᛶ㸪ග࢔ࣞࣝࢠ࣮ᛶ㸪ග㑇ఏẘᛶࡲࡓࡣගࡀࢇཎᛶࢆㄏᑟࡍࡿྍ⬟ᛶࡀ࠶ࡿࡶࡢࡢ㸪ICH ࡟࠾࠸࡚సᡂ
ࡉࢀࡓ་⸆ရࡢගᏳ඲ᛶホ౯࡟㛵ࡍࡿ࢞࢖ࢻࣛ࢖ࣥࡢ㐺⏝⠊ᅖࡣ㸪ග่⃭ᛶ࡜ග࢔ࣞࣝࢠ
࣮ᛶ࡟㝈ᐃࡋ࡚࠸ࡿ㸬ගᏳ඲ᛶホ౯࡟㛵ࡋ࡚ࡣࡇࢀࡲ࡛ 2002 ᖺ࡟Ḣᕞ་⸆ရᑂᰝᗇ
(EMEA; ⌧ EMA)/Ḣᕞ་⸆ရጤဨ఍ (CPMP)㸪⩣ᖺࡢ 2003 ᖺ࡟ࡣ⡿ᅜ㣗ရ་⸆ရᒁ
(FDA)/་⸆ရホ౯◊✲ࢭࣥࢱ࣮(CDER)㸪ࡉࡽ࡟ 2004 ᖺ࡟ࡣ⤒῭༠ຊ㛤Ⓨᶵᵓ (OECD)࠿
ࡽホ౯᪉ἲࡀᥦ♧ࡉࢀ࡚࠸ࡿࡀ㸪ࡇࢀࡽ 3 ࡘࡢ࢞࢖ࢻࣛ࢖ࣥ/࢞࢖ࢲࣥࢫࡢඹ㏻Ⅼ࡜ࡋ࡚㸪໬ྜ≀ࡢග໬Ꮫⓗ≉ᛶࢆホ౯ࡍࡿᚲせᛶࡀグ㏙ࡉࢀ࡚࠸ࡿࡇ࡜ࡀᣲࡆࡽࢀࡿ㸬ࡇࢀࡽࡢ࢞
࢖ࢻࣛ࢖ࣥ/࢞࢖ࢲࣥࢫ࡛ࡣึᮇࢫࢡ࣮ࣜࢽࣥࢢ࡜ࡋ࡚ UV/VIS ྾཰≉ᛶࡀᥦ᱌ࡉࢀ࡚࠸
ࡿ㸬OECD ࢞࢖ࢻࣛ࢖࡛ࣥࡣホ౯⣔࡟࠾ࡅࡿࣔࣝ྾ගಀᩘ࡟㛵ࡍࡿ㜈್ࢆ 10 M-1㺃cm-1 ࡜タᐃࡋ࡚࠾ࡾ㸪ᐇ㉁ⓗ࡟ࡣ඲࡚ࡢ໬ྜ≀ࡀࡇࡢ㜈್ࢆ㉸࠼ࡿ㸬ࡑࢀᨾ㸪ࡼࡾ⌧ᐇⓗ࡞㜈್ࡢタᐃࢆ┠ⓗ࡜ࡋࡓ◊✲ࡀ⾜ࢃࢀ㸪Henry ࡽࡣ᪤▱ගẘᛶ་⸆ရࢆᑐ㇟࡜ࡋ࡚ග྾཰≉ᛶࢆ⢭ᰝࡋࡓ⤖ᯝ㸪⤒㦂๎ⓗ࡟ࣔࣝ྾ගಀᩘ 1,000 M-1㺃cm-1 ࡀࡼࡾ㐺ษ࡞㜈್࡛࠶ࡾ㸪ࡇࢀࢆ㉸࠼࡞࠸་⸆ရ࡟ࡘ࠸࡚ࡣගᏳ඲ᛶホ౯ࢆᐇ᪋ࡍࡿᚲせᛶࡣప࠸ࡇ࡜ࢆ♧ࡋ࡚࠸ࡿ(Henry et al., 2009)㸬EMA ࡣ 2010 ᖺ࡟⮬ࡽࡢ࢞࢖ࢲࣥࢫ࡟㛵ࡍࡿ Question and Answers ࢆබ⾲ࡋ㸪ࡇࡢ⪃࠼ࢆ㏣㝶ࡋ࡚࠸ࡿ㸬 ICH ࡟࠾࠸࡚་⸆ရࡢගᏳ඲ᛶホ౯ࡀᏳ඲ᛶ࡟㛵ࡍࡿࢺࣆࢵࢡ࡜ࡋ࡚ 2010 ᖺ࡟᥇ᢥࡉࢀ㸪EWG ࡀⓎ㊊ࡋ㸪3 ᖺ㛫ࡢ᳨ウᮇ㛫ࢆ⤒࡚㸪2013 ᖺ 11 ᭶࡟ගᏳ඲ᛶホ౯࡟㛵ࡍࡿ࢞࢖ࢻࣛ࢖ࣥࡢ Step 4 ᩥ᭩ࡀྜពࡉࢀࡓ㸬ICH ࢞࢖ࢻࣛ࢖ࣥ࡟࠾
࠸࡚ගẘᛶホ౯ࡢせྰࢆุ᩿ࡍࡿึᮇホ౯࡟࠾࠸࡚㸪ࣔࣝ྾ගಀᩘࡀ 1000 M-1㺃cm-1 ࢆ㉸࠼ࡿ≀㉁࡛࠶ࡗ࡚ࡶ໬Ꮫⓗග཯ᛂᛶホ౯⣔࡟࠾࠸࡚㝜ᛶ࡛࠶ࢀࡤ㸪ࡉࡽ࡞ࡿගẘᛶホ౯ࡸ⮫ᗋヨ㦂࡟࠾ࡅࡿග㜵ᚚᥐ⨨ࡣ୙せ࡜ࡉࢀ࡚࠸ࡿ㸬ROS ࢔ࢵࢭ࢖ࡣ໬Ꮫⓗග཯ᛂᛶ࠿ࡽ⿕㦂≀㉁ࡢගẘᛶࡀ㝜ᛶ࡛࠶ࡿࡇ࡜ࢆ☜ㄆ࡛ࡁࡿ᪉ἲ࡜ࡋ࡚㸪་⸆ရࡢගᏳ඲ᛶホ౯࡟฼⏝ࡉ
ࢀ࡚࠸ࡿ㸬

9
15

⾲ 1

Ḣ⡿ࡢගᏳ඲ᛶホ౯࢞࢖ࢻࣛ࢖ࣥ
EMEA/CPMP࢞࢖ࢻࣛ࢖ࣥ

Guidance on
photosafety
testing

FDA/CDER
Guidance forindustry
photosafety
testingOECD

ICH

In vitro 3T3 NRU
phototoxicity test

Photosafety
evaluation ofpharmaceuticals(S10)ගẘᛶホ౯ࡀᚲせ࡜࡞ࡿ᮲௳࣭໬Ꮫ≀㉁඲⯡ ࣭඲࡚ࡢཎ⸆࠾ࡼ ࣭UV/VIS ྾཰ࢆ ࣭᪂つ་⸆ရ᭷࣭ࣂ࢖࢜ࢸࢡࣀࣟ ࡧ〇๣ᡂศ♧ࡍ໬Ꮫ≀㉁ຠᡂศ(API)ࢪ࣮་⸆ရ࣭እ⏝๣ࡣ〇๣࡛ ඲⯡࣭᪂つῧຍ๣
ࡢᐇ᪋ࢆ᥎ዡ
࣭⤒⓶ᢞ୚⏝⮫
ᑐ㇟໬ྜ≀ࡢ⠊ᅖᗋ〇๣㸦⓶⭵㈞
௜๣࡞࡝㸧࣭ග⥺ຊᏛ⒪ἲ
⏝〇๣
࣭290–700 nm ࣔ ࣭290–700 nm ࣔ ࣭290–700 nm ࣔ ࣭290–700 nm ࣔࣝ྾ගಀᩘ࡟
ࣝ྾ගಀᩘ࡟
ࣝ྾ගಀᩘ㸸10 ࣝ྾ගಀᩘ㸸1000 M-1㺃cm-1
㛵ࡍࡿグ㍕࡞
㛵ࡍࡿグ㍕࡞
M-1㺃cm-1 ௨ୖ௨ୖࡋࡋ͌UV/VIS ࡢ྾཰
UV/VIS ྾཰≉ᛶ࡟ࡼࡾ㸪཯ᛂᛶ
࡟ᐩࢇࡔศᏊ
✀ࢆᙧᡂࡍࡿ㸦ROS ࢔ࢵࢭ࢖㸧࣭ᒁᡤ㐺⏝࣭⓶⭵࣭║࡟㐺⏝࣭⓶⭵㺃║࡟༑ศ
ᢞ୚⤒㊰࡜యෆศ ࣭⓶⭵㺃║࡟฿㐩ࡶࡋࡃࡣศᕸ
㔞ศᕸ㐺⏝እᕸ࣭⓶⭵㺃║࡟ᙳ㡪ࢆ୚࠼ࡿࡶࡢ

ホ౯᪉ἲ

Photoirritation

Photoallergy

Photogenotoxicity

࣭3T3 NRU
࣭3T3 NRU
࣭3T3 NRU
࣭3T3 NRU
phototoxicity test phototoxicity test phototoxicity test phototoxicity test࣭࣐࢘ࢫ㸪ࣔࣝࣔ
ࢵࢺ㸪ࣈࢱ㸪࢘ࢧࢠࢆ⏝࠸ࡓ
in vivo ヨ㦂࣭ᨵⰋࡉࢀࡓ
࣭⤒⓶ᢞ୚་⸆ရ
LLNA ࢔ࢵࢭ
࡟࠾࠸࡚ࡶ㸪㠀
࢖ࡸ MEST ࢔⮫ᗋග࢔ࣞࣝ
ࢵࢭ࢖ࡀᑗ᮶㐺⏝እ㐺⏝እࢠ࣮ᛶヨ㦂ࡢ
ⓗ࡟᭷⏝࡟࡞
⤖ᯝࡢࣄࢺ࡬
ࡿྍ⬟ᛶࢆ♧
ࡢண ᛶࡣ୙
၀ࡍࡿࡢࡳ᫂࡜ࡋ࡚࠸ࡿ
࣭ග໬ᏛⓗᰁⰍయ ࣭ࢧࣝࣔࢿࣛ⳦㸪
␗ᖖㄏⓎᛶヨ㓝ẕ㸪V79 ⣽⬊ࢆ⏝࠸ࡓ in㦂࣭in vivo ࢥ࣓ࢵࢺ vitro ග㑇ఏẘ
㐺⏝እ
㐺⏝እ
ヨ㦂
ᛶヨ㦂
࣭ࢺࣛࣥࢫࢪ࢙ࢽ
ࢵࢡኚ␗ཎᛶ
ࣔࢹࣝ

1016

2 ヨ㦂ࡢࣉࣟࢺࢥࣝ࠾ࡼࡧุᐃᇶ‽
2.1

ᐃཎ⌮

 ஦๓ࡢ᳨ウ࡟࠾࠸࡚㸪ග↷ᑕࡋࡓ⸆≀࠿ࡽ⏘⏕ࡉࢀࡿ singlet oxygen ࡜ superoxide ࡢ 2✀ࡢ ROSᐃࡀ⢭ᗘࡢ㧗࠸ගᏳ඲ᛶホ౯࡟ᐤ୚ࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓ (Onoue et al.,

2006&2008)㸬ࡑࡇ࡛㸪ࡇࡢ 2 ✀ࡢ ROS ⏘⏕ࢆホ౯ࡍࡿࡇ࡜ࢆ┠ⓗ࡜ࡋ࡚ ROS ࢔ࢵࢭ࢖ࡀᵓ⠏ࡉࢀࡓ㸬Singlet oxygen ࡣ p-nitrosodimethyl aniline ࡢኚⰍࢆᣦᶆ࡜ࡋ࡚ࡑࡢ⏘⏕ࢆホ౯࡛ࡁࡿ㸬ࡍ࡞ࢃࡕ㸪ບ㉳໬ྜ≀࠿ࡽ⏘⏕ࡉࢀࡓ singlet oxygen ࡣ཯ᛂᾮ୰ࡢ imidazole ⎔࡜཯ᛂࡋ࡚ trans-annular peroxide intermediate ࢆᙧᡂࡍࡿࡀ㸪ࡇࢀࡀ p-nitrosodimethyl anilineࢆ㓟໬ࡍࡿࡇ࡜࡛ኚⰍࢆࡶࡓࡽࡋ㸪440 nm ࡢ྾ග್ࡀῶᑡࡍࡿ㸬ࡇࡢ྾ග್ࡢῶᑡࡀ
singlet oxygen ࡢ⏕ᡂࢆ♧၀ࡍࡿࡶࡢ࡛࠶ࡿ㸬
Photoirradiated chemical + dissolved oxygen Ѝoxidized chemical + singlet oxygen
singlet oxygen + imidazole ĺ [peroxide intermediate] ĺ oxidized imidazole[peroxide intermediate] + p-nitrosodimethyl aniline ĺ p-nitrosodimethyl aniline + products
 Superoxide ࡶྠࡌࡃศගᏛⓗᡭἲ࡟ࡼࡗ࡚

ᐃࡀྍ⬟࡛࠶ࡾ㸪ᇶ㉁࡜ࡋ࡚ nitroblue

tetrazolium (NBT) ࢆ ౑⏝ ࡍࡿ 㸬ບ㉳ ໬ྜ ≀࠿ࡽ ⏘⏕ ࡉࢀࡿ superoxide ࡣ one-electrontransfer reaction ࡟ࡼࡾ NBT ࢆ㑏ඖࡋ㸪⏕ᡂࡉࢀࡿ monoformazan (NBT+) ࡣ 560 nm ࡟
UV ྾཰ࢆ᭷ࡍࡿ㸬ࡇࡢ྾ග್ࡢୖ᪼ࡀ superoxide ࡢ⏕ᡂࢆ♧၀ࡍࡿ㸬
Photoirradiated chemical + dissolved oxygen Ѝoxidized chemical + superoxide
superoxide + NBT ĺ O2 + NBT+
 ࡇࢀࡽࡢ཯ᛂࡣ photodynamic ࡟ಁ㐍ࡉࢀࡿࡀ㸪ග↷ᑕᙉᗘ㸪ග↷ᑕ᫬㛫㸪ࢧࣥࣉࣝᗘ㸪ඹᏑࡍࡿ᭷ᶵ⁐፹ࢆࡣࡌࡵ࡜ࡍࡿ✀ࠎࡢ࢔ࢵࢭ࢖᮲௳࡟ࡼࡗ࡚ࡶ኱ࡁࡃኚືࡀㄏⓎࡉࢀࡿࡓࡵ㸪⣽ᚰࡢὀពࡀᚲせ࡛࠶ࡿ㸬

2.2ᮦᩱ࠾ࡼࡧヨ⸆

2.2.1

ROS ࢔ࢵࢭ࢖⏝ヨ⸆㢮࡞ࡽࡧ࡟ࡑࡢㄪ〇1117

 NaH2PO4࣭2H2O (CAS#13472-35-0), Na2HPO4࣭12H2O (CAS#10039-32-4), dimethyl sulfoxide(DMSO,

CAS#67-68-5),

p-nitrosodimethyl

aniline(RNO,

CAS#138-89-6),

imidazole(CAS#288-32-4), and nitroblue tetrazolium (NBT, CAS#298-83-9) ࡣᕷ㈍ࡢヨ⸆ࢆ౑⏝ࡍࡿ.

2.2.2 20 mM sodium phosphate buffer (NaPB, pH7.4)
 3.8 mmol (593 mg) ࡢ NaH2PO4࣭2H2O ࡜㸪16.2 mmol (5.8 g) ࡢ Na2HPO4࣭12H2O ࢆ㔞ࡾྲྀࡾ㸪900 mL ࡢ⢭〇Ỉ࡟⁐ゎࡋࡓ㸬HCl ࡟ࡼࡗ࡚ pH7.4 ࡟ㄪᩚࡋ㸪⢭〇Ỉ࡛ 1 L ࡟࣓ࢫ࢔ࢵࣉࡋ࡚ࡼࡃΰ࿴ࡍࡿ㸬

2.2.3 200 PM p-nitrosodimethyl aniline Ỉ⁐ᾮ 20 μmol (3 mg) ࡢ p-nitrosodimethyl aniline ࢆ 100 mL ࡢ NaPB ࡟⁐ゎࡍࡿ㸬ㄪ〇ᚋࡣ㐽ගࣂ࢖࢔ࣝ࡟ධࢀ㸪෭ⶶᗜ࡟࡚ಖ⟶ࡍࡿ㸬

2.2.4

200 PM imidazole Ỉ⁐ᾮ 200 μmol (13.6 mg) ࡢ imidazole ࢆ 10 mL ࡢ NaPB ࡟⁐ゎࡋ㸪ࡉࡽ࡟ NaPB ࡛ 100 ಸᕼ㔘ࡍࡿ㸬ㄪ〇ᚋࡣ㐽ගࣂ࢖࢔ࣝ࡟ධࢀ㸪෭ⶶᗜ࡟࡚ಖ⟶ࡍࡿ㸬

2.2.5 400 PM NBT Ỉ⁐ᾮ 40 μmol (32.7 mg) ࡢ NBT ࢆ 100 mL ࡢ NaPB ࡛⁐ゎࡋࡓ㸬NBT ࡣ⁐ゎᗘࡀప࠸ࡓࡵ㸪㉸㡢Ἴฎ⌮ࡸ࣎ࣝࢸࢵࢡࢫ➼ࡢ⁐ゎ⿵ຓ᧯సࡀᚲせ࡜࡞ࡿࡇ࡜ࡀ࠶ࡿ㸬ㄪ〇ᚋࡣ㐽ගࣂ࢖࢔ࣝ࡟ධࢀ㸪෭ⶶᗜ࡟࡚ಖ⟶ࡍࡿ㸬

2.3

⿕㦂≀㉁

 ROS ࢔ࢵࢭ࢖ࢆᐇ᪋ࡍࡿ㝿࡟ࡣ㸪 DMSO ࡛ 10 mM ࡢ stock solution ࢆ࢔ࢵࢭ࢖┤๓࡟ㄪ〇ࡋ㸪ROS ࢔ࢵࢭ࢖࡟࠾ࡅࡿ⤊⃰ᗘࡣ 200 μM ࡜ࡍࡿ㸬ගᏳᐃᛶ࡟ὀពࢆࡋࡘࡘ㸪࣎ࣝࢸࢵࢡࢫ࣑࢟ࢧ࣮࡟࡚ 5–10 ศ⛬ᗘΰ࿴ࡋ⁐ゎᛶࢆ☜ㄆࡍࡿࡇ࡜ࢆ᥎ዡࡍࡿࡀ㸪⿕㦂≀㉁ࡢ⁐ゎᗘࡀప࠸ሙྜ࡟ࡣ NaPB ࡛⁐ゎࡋ࡚ࡶࡼ࠸㸬ࡑࢀ࡛ࡶ࡞࠾⁐ゎࡋ࡞࠸ሙྜ࡟ࡣ㸪DMSO stock solution ࢆ DMSO ࡛ᕼ㔘ࡋ⁐ゎᛶࢆ☜ㄆࡋ࡚ ROS ࢔ࢵࢭ࢖ࢆᐇ᪋ࡍࡿࡇ࡜
࡜ࡍࡿ㸬ᕼ㔘ࡍࡿሙྜ࡟ࡣ ROS ࢔ࢵࢭ࢖࡟࠾ࡅࡿ⤊⃰ᗘࡀ㸪20㸪50㸪࠶ࡿ࠸ࡣ 100 μM ࡜࡞ࡿࡇ࡜ࡀᮃࡲࡋ࠸ࡀࡇࡢ㝈ࡾ࡛ࡣ࡞࠸㸬ᕼ㔘ࡋࡓሙྜ࡟ࡣุᐃᇶ‽ࡀ㠀ᕼ㔘᫬࡜ኚࢃࡿ
ࡢ࡛ὀពࡀᚲせ࡛࠶ࡿ㸬࡞࠾㸪200 μM ࡢ⤖ᯝࡢุᐃࢆ⏝࠸ࡿࡇ࡜ࡀ᥎ዡࡉࢀ㸪⁐ࡅ࡞࠸ሙྜ࡟ࡣప⃰ᗘࢆ౑⏝ࡍࡿࡀ㸪⤖ᯝࡢุ᩿࡟ࡣὀពࡀᚲせ࡛࠶ࡿ㸬

12
18

2.4

㝧ᛶ࡞ࡽࡧ࡟㝜ᛶᑐ↷≀㉁ ROS ࢔ࢵࢭ࢖࡟࠾࠸࡚ࡣ㝧ᛶᑐ↷≀㉁࡜ࡋ࡚ quinine HCl (CAS#6119-47-7)㸪ࡑࡋ࡚㝜ᛶᑐ↷≀㉁࡜ࡋ࡚ sulisobenzone (CAS#4065-45-6) ࢆ⏝࠸ࡿࡇ࡜ࡀᮃࡲࡋ࠸㸬࠸ࡎࢀࡶ DMSO
stock solution ࢆ࢔ࢵࢭ࢖┤๓࡟ㄪ〇ࡍࡿࡇ࡜࡜ࡍࡿ㸬

2.5

ჾල࣭ᶵჾ

2.5.1

Solar simulator ග⥺㐣ᩄ⑕ࡢ࡯࡜ࢇ࡝ࡀኴ㝧ග࡬ࡢ᭚㟢࡟ࡼࡗ࡚ច㉳ࡉࢀࡿࡇ࡜ࢆ⪃៖ࡍࢀࡤ㸪ROS ࢔ࢵࢭ࢖࡟౑⏝ࡍࡿ solar simulator ࡣྍ⬟࡞㝈ࡾ CIE85/1989 ᶆ‽᫨ග࡟㏆࠸ࡇ࡜ࡀᮃࡲࡋ
࠸㸬౛࠼ࡤ㸪ග※࡜ࡋ࡚ Xe ࣛࣥࣉ (1,500 W) ࢆ⿦ഛࡋࡓ Atlas Suntest CPS/CPS plus (Atlas
Material Technology LLC, Chicago, USA) ࠶ࡿ࠸ࡣ Seric SXL-2500V2 (SERIC., Ltd., Tokyo,
Japan) ࢆ౑⏝ࡋ㸪ᶆ‽᫨ග࡟ࡼࡾ㏆ࡃࡍࡿ࡭ࡃ 290 nm ௨ୗࡢ↷ᑕගࢆ࢝ࢵࢺࡍࡿࡓࡵ࡟
UV ࣇ࢕ࣝࢱ࣮ࢆ⿦╔ࡋ࡚⏝࠸ࡿ㸬UVA detector Dr. Hönle #0037 (Dr. Hönle, München,
Germany) ࡛↷ᑕගࢆࣕࣥࣂ࣮ෆࡢ

ᐃࡋࡓ࡜ࡁ㸪⣙ 2 mW/cm2 ࡢ↷ᑕᙉᗘ࡛࠶ࡾ㸪solar simulator ࡢࢳᗘࡣ 20–29Υ ࡢ⠊ᅖ࡟ไᚚࡉࢀ࡚࠸ࡿࡇ࡜ࡀᮃࡲࡋ࠸㸬ࡲࡓ㸪solar simulator࡟ࡼࡗ࡚ࡣ↷ᑕ࣒ࣛࡀⓎ⏕ࡍࡿࡇ࡜ࡀ࠶ࡿࡢ࡛㸪࢔ࢵࢭ࢖ࡢ๓࡟᳨ドࡋ㸪↷ᑕ࣒ࣛࡀ࡞࠸
㡿ᇦࢆ᫂☜࡟ࡋ࡚࠾ࡃᚲせࡀ࠶ࡿ㸬ᮏ✏࡛ᥦ♧ࡍࡿࢹ࣮ࢱࡣࡍ࡭࡚ࡇࢀࡽࡢ↷ᑕ⿦⨨ࢆ⏝࠸࡚ ROS ࢔ࢵࢭ࢖ࢆᐇ᪋ࡋࡓࡶࡢ࡛࠶ࡿ㸬௚ࡢ solar simulator ࢆ⏝࠸࡚౑⏝ࡍࡿࡇ࡜ࡶྍ
⬟࡛࠶ࡿࡀ㸪ࡑࡢሙྜ࡟ࡣ㝧ᛶᑐ↷㸦quinine㸧㸪㝜ᛶᑐ↷㸦sulisobenzone㸧࡞ࡽࡧ࡟᥎ዡ໬
ྜ≀ࢭࢵࢺ㸦recommended chemical set㸧ࢆ⏝࠸࡚᥎ዡࣉࣟࢺࢥ࣮ࣝ࡟グ㍕ࡉࢀ࡚࠸ࡿ᮲௳᭱㐺໬ࢆᚲせ࡜ࡍࡿ㸬

2.5.2

ROS࢔ࢵࢭ࢖⏝reaction container ᨃఝኴ㝧ගࢆ↷ᑕࡍࡿ㝿࡟ࡣࢧࣥࣉࣝࢆ 96 well microplate (ᖹᗏ㸪ࢥ࣮ࢸ࢕ࣥࢢ࡞ࡋ) ࡟ධࢀ࡚⾜࠺ࡀ㸪ࡑࡢ㝿࡟ࡣỈศ᥹Ⓨ࡟ࡼࡿࢧࣥࣉࣝ⃰ᗘኚືࢆᅇ㑊ࡍࡿࡓࡵ࡟ࣇࢱࢆࡍࡿ
ࡇ࡜ࡀᚲせ࡛࠶ࡿ㸬㏻ᖖࡢ microplate ࡢࣇࢱ࡛ࡣ↷ᑕගࡢࢫ࣌ࢡࢺࣝࣃࢱ࣮ࣥࡀኚ໬ࡍࡿ
ྍ⬟ᛶࡀ㧗ࡃ㸪ࡑࢀᨾ solar simulator ࠿ࡽࡢ↷ᑕග࡟ᙳ㡪ࡀฟ࡞࠸ࡼ࠺␃ពࡍ࡭ࡁ࡛࠶ࡿ㸬ᮃࡲࡋࡃࡣᅗ 2 㸦ḟ㡫㸧࡟♧ࡍ quartz reaction container (࢜ࢨ࣡⛉Ꮫ㸪ឡ▱) ࢆ⏝࠸ࡿࡇ࡜ࡀ᥎ዡࡉࢀࡿࡀ㸪ࡇࢀ࡟㝈ᐃࡉࢀࡿࡶࡢ࡛ࡣ࡞ࡃྠ➼ရࢆ⏝࠸࡚ࡶࡼ࠸㸬᥎ዡࡉࢀࡿ quartz
reaction container ࡣࢫࢳ࣮ࣝ〇ࡢᅛᐃල㸪ⓑⰍࢸࣇࣟࣥࢩ࣮ࢺ㸪ࡑࡋ࡚ࣇࢱࡢᙺ┠ࢆᯝࡓࡍquartz plate ࠿ࡽᵓᡂࡉࢀࡿ㸬ࡲࡓ㸪Onoue ࡽࡣඛ࡟㸪96 well ࡢ࠺ࡕᅗ 2 ࡟♧ࡍ㡿ᇦ (B2–
G11) ࡟ࡣ࡯ࡰྠ➼ࡢ↷ᑕ㔞ࡢᨃఝኴ㝧ගࡀὀࡀࢀࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋ࡚࠾ࡾ (Onoue et al.,13
19

2008)㸪ࡑࢀᨾ㸪ಙ㢗࠶ࡿࢹ࣮ࢱࢆᚓࡿࡓࡵ࡟ࡣࡇࡢ㡿ᇦࢆ⏝࠸࡚ ROS ࢔ࢵࢭ࢖ࢆ⾜࠺ࡇ࡜ࡀ᥎ዡࡉࢀࡿ㸬

2.5.3

ࡑࡢ௚࡟ᚲせ࡜ࡉࢀࡿჾල࣭⿦⨨ ROS ࢔ࢵࢭ࢖ࢆᐇ᪋ࡍࡿ㝿࡟ࡣඛ㏙ࡢ⿦⨨࣭ჾලࡢ࡯࠿㸪ᰯṇࡉࢀࡓ UVA detector㸪ᗘィ㸪࣐࢖ࢡࣟࣉ࣮ࣞࢺ࣮ࣜࢲ࣮㸪ගᏛ㢧ᚤ㙾㸪࣎ࣝࢸࢵࢡࢫ࣑࢟ࢧ࣮㸪ࣉ࣮ࣞࢺࢩ࢙࣮
࣮࢝㸪㉸㡢ἼὙίᶵ㸪࣐࢖ࢡࣟࣆ࣌ࢵࢺ࡞࡝ࡀᚲせ࡛࠶ࡿ㸬

ᅗ 2

2.6

reaction container ࡢᵓᡂ

ヨ㦂᪉ἲ࡞ࡽࡧ࡟ࢹ࣮ࢱゎᯒ ROS ࢔ࢵࢭ࢖࡛ࡣ singlet oxygen ࡜ superoxide ࡢ཮᪉ࢆศᯒࡍࡿࡓࡵ㸪ࡲࡎࡑࢀࡒࢀࡢᐃ࡟ᚲせ࡞ assay mixture ࢆᅗ 3 ࡟ᚑ࠸సᡂࡍࡿ㸬singlet oxygenᐃ⏝ࡢ assay mixture(1 mL) ࡟ࡣ௵ពࡢ⃰ᗘࡢ⿕㦂≀㉁㸪50 n mol ࡢ p-nitrosodimethyl aniline㸪50 n mol ࡢ
imidazole ࡀࡑࢀࡒࢀྵࡲࢀ㸪superoxideᐃ⏝ࡢ assay mixture (1 mL) ࡣ௵ពࡢ⃰ᗘࡢ⿕㦂≀㉁࡜ 50 n mol ࡢ NBT ࢆྵ᭷ࡍࡿ㸬ㄪ〇ᚋ㸪200 μL ࡢ assay mixture ࢆ 96 well
microplate ࡟ n=3 ࡟࡞ࡿࡼ࠺ᅗ 4 ࡟ᚑࡗ࡚⛣ࡋ㸪ࡑࡢᚋ࡟ගᏛ㢧ᚤ㙾ほᐹ (100) ࡟ࡼࡗ࡚୙⁐ᛶ⢏Ꮚ࠶ࡿ࠸ࡣᯒฟ≀ࡢ᭷↓ࢆ☜ㄆࡍࡿ㸬ࡶࡋ㸪⿕㦂≀㉁ࡢ⁐ゎᗘࡢၥ㢟࡛᏶඲࡟⁐ゎࡋ࡚࠸࡞࠿ࡗࡓሙྜ࡟ࡣ DMSO stock solution ࢆᕼ㔘ࡋ࡚ 200μM ࡼࡾప࠸⃰ᗘ࡛ࡢ
ROS ࢔ࢵࢭ࢖ࢆᐇ᪋ࡍࡿࡇ࡜࡛ᑐᛂࡍࡿ㸬㝧ᛶ࡞ࡽࡧ࡟㝜ᛶࢥࣥࢺ࣮ࣟࣝ࡜ࡋ࡚ quinine

1420

(200 μM) ࡜ sulisobenzone (200 μM) ࡢ assay mixture ࢆㄪ〇ࡍࡿ࡜࡜ࡶ࡟㸪blank ࡜ࡋ࡚⿕㦂≀㉁ࡢ stock solution ࡢ௦ࢃࡾ࡟ NaPB ࢆຍ࠼ࡓࡶࡢࡶేࡏ࡚⏝ពࡍࡿ㸬Plate shaker ࡟ࡼࡗ࡚ 5 ⛊⛬ᗘ᧠ᢾࡋࡓᚋ㸪ᨃఝኴ㝧ග↷ᑕ๓ࡢྛ assay mixture ࡢ྾ග್ (singlet oxygenࡣ 440 nm, superoxide ࡣ 560 nm) ࢆࣉ࣮ࣞࢺ࣮ࣜࢲ࣮࡛

ᐃࡍࡿ㸬96 well microplate ࢆquartz reaction container ࡟ࢭࢵࢺࡋ㸪Ẽᐦᛶࢆ㧗ࡵࡿࡓࡵ࡟ quartz plate ࢆࡋࡗ࠿ࡾ࡜ᅛᐃ
ࡍࡿ㸬Solar simulator ࡢࢳࣕࣥࣂ࣮ෆ࡟ assay mixture ࢆධࢀࡿ࠿㸪↷ᑕ࢚ࣜ࢔࡟ assay
mixture ࢆ㓄⨨ࡋ㸪1 ᫬㛫ᨃఝኴ㝧ගࢆ↷ᑕࡍࡿ㸦↷ᑕ㔞㸸Suntest CPS/CPS+ࡣ⣙ 2.0 mW/cm2㸪
SXL-2500V2 ࡣ 3.0̽5.0 mW/cm2㸧㸬㟢ගᚋ࡟ࡣ⿕㦂≀㉁ࡸ NBT+ ࡀᯒฟࡋ࡚࠸ࡿࡇ࡜ࡀ࠶
ࡿࡓࡵ㸪 1 ศ⛬ᗘ plate shaker ࡛ΰ࿴ࡋ㸪ࡑࡢᚋ࡟ࣉ࣮ࣞࢺ࣮ࣜࢲ࣮࡟࡚ྛ assay mixtureࡢ྾ග್ࢆྠᵝ࡟

ᐃࡍࡿ㸬

15
21

ᅗ 3

ROS ࢔ࢵࢭ࢖ࡢ࣮࣡ࢡࣇ࣮ࣟ

Blank ࡞ࡽࡧ࡟⿕㦂≀㉁ࡢ㟢ග๓ᚋࡢ྾ග್࠿ࡽ singlet oxygen ࡞ࡽࡧ࡟ superoxide ࡢ⏘⏕ࢆ௨ୗࡢ㏻ࡾࡑࢀࡒࢀィ⟬ࡍࡿ㸬⿕㦂≀㉁࡟╔Ⰽࡀㄆࡵࡽࢀ㸪࡞࠾࠿ࡘග↷ᑕ࡟ࡼࡗ࡚ኚⰍࡀㄆࡵࡽࢀࡿሙྜ࡟ࡣ㸪ROS ཯ᛂᇶ㉁ࢆྵࡲ࡞࠸ Assay mixture ࢆ⏝ពࡋ࡚ ROS

16
22

assay ࡜ྠᵝࡢ᧯సࢆ⾜࠸㸪440 nm ࠶ࡿ࠸ࡣ 560 nm ࡟࠾ࡅࡿ྾ග್ኚ໬ࢆ ROS assay ࢹ࣮ࢱ࠿ࡽᕪࡋᘬࡃࡇ࡜࡛ᑐᛂࡀྍ⬟࡛࠶ࡿ㸬
Singlet oxygen ࡢ⏘⏕㸸'A440×103 = {A440(-) – A440(+) – (A – B)}103
A440(-): 㟢ග๓ࡢ assay mixture ࡢ 440 nm ࡟࠾ࡅࡿ྾ග್
A440(+): 㟢ගᚋࡢ assay mixture ࡢ 440 nm ࡟࠾ࡅࡿ྾ග್
A:

㟢ග๓ࡢ blank ࡢ 440 nm ࡟࠾ࡅࡿ྾ග್

B:

㟢ගᚋࡢ blank ࡢ 440 nm ࡟࠾ࡅࡿ྾ග್

Superoxide ࡢ⏘⏕㸸'A560×103 = {A560(+) – A560(-) – (B – A)}103
A560(-): 㟢ග๓ࡢ assay mixture ࡢ 560 nm ࡟࠾ࡅࡿ྾ග್
A560(+): 㟢ගᚋࡢ assay mixture ࡢ 560 nm ࡟࠾ࡅࡿ྾ග್

ᅗ 4

2.7

A:

㟢ග๓ࡢ blank ࡢ 560 nm ࡟࠾ࡅࡿ྾ග್

B:

㟢ගᚋࡢ blank ࡢ 560 nm ࡟࠾ࡅࡿ྾ග್

ROS ࢔ࢵࢭ࢖࡟࠾ࡅࡿࢧࣥࣉࣝࣞ࢖࢔࢘ࢺ

ࢹ࣮ࢱ᥇⏝᮲௳

 ROS ࢔ࢵࢭ࢖ࢆ⾜࠺㝿࡟ࡣ௨ୗࡢ᮲௳ࢆ‶ࡓࡋࡓሙྜ࡟ࡢࡳࢹ࣮ࢱࢆ᥇⏝ࡍࡿࡶࡢ࡜ࡍ
ࡿ㸬

17
23

(i) 㟢ග๓ࡢ assay mixture ୰࡟࠾࠸࡚⿕㦂≀㉁ࡀ⁐ゎࡋ࡚࠸ࡿࡇ࡜
(ii) blank㸪㝧ᛶ࡞ࡽࡧ࡟㝜ᛶᶆ‽≀㉁࡟ࡘ࠸࡚ࡶྠ᫬࡟ ROS ࢔ࢵࢭ࢖࡛ホ౯ࡋ࡚࠸ࡿࡇ࡜㸬ࡑࡢ㝿࡟ࡣ㝧ᛶᶆ‽≀㉁ quinine (200 μM) ࡢ singlet oxygen ࢹ࣮ࢱࡀ 300 ௨ୖ㸪superoxide
ࡀ 200 ௨ୖ࡛࠶ࡾ㸪㝜ᛶᶆ‽≀㉁ sulisobenzone (200 μM) ࡢ singlet oxygen ࢹ࣮ࢱࡀ 25 ௨ୗ㸪superoxide ࡀ 20 ௨ୗ࡛࠶ࡿࡇ࡜㸬(iii) ᶆ‽≀㉁࡞ࡽࡧ࡟⿕㦂≀㉁ࡢ assay mixture ࡢ 440 nm ࠶ࡿ࠸ࡣ 560 nm ࡟࠾ࡅࡿ྾
ග್ࡀ 0.02 ࠿ࡽ 1.5 ࡢ⠊ᅖ࡟ධࡗ࡚࠸ࡿࡇ࡜㸬

2.8

㝧ᛶᇶ‽

 ⣙ 40 ✀ࡢᕷ㈍་⸆ရ໬ྜ≀࡟࠾ࡅࡿ ROS ࢔ࢵࢭ࢖ࢹ࣮ࢱ࡜ࡑࢀࡽࡢගᏳ඲ᛶ࡟㛵ࡍࡿ⮫ᗋ᝟ሗ࠿ࡽ㸪ROS ࢔ࢵࢭ࢖࡟࠾ࡅࡿ⤒㦂๎ⓗ࡞㜈್ࢆタᐃࡋࡓ (Onoue et al., 2008)㸬
⿕㦂≀㉁ (200 μM) ࡢ ROS data ࡟ࡘ࠸࡚㸪singlet oxygen ('A440 nm࣭103): 25 ௨ୖ㸪ࡑࡋ࡚superoxide ('A560 nm࣭103): 20 ௨ୖࡢ࠸ࡎࢀ࠿ࡢ᮲௳ࢆ‶ࡓࡋࡓ࡜ࡁ㸪⿕㦂≀㉁ࡣගឤཷᛶ
ࡀ㧗ࡃ࡞ࢇࡽ࠿ࡢගẘᛶ཯ᛂࢆច㉳ࡍࡿྍ⬟ᛶࡀ࠶ࡿࡶࡢ࡜ุ᩿࡛ࡁࡿ㸬ࡓࡔࡋ㸪⁐ゎᗘࡢၥ㢟࡛ᕼ㔘ୗ࡛ࡢ ROS ࢔ࢵࢭ࢖ࢆవ൤࡞ࡃࡉࢀࡓ㝿࡟ࡣࡇࡢ㝈ࡾ࡛ࡣ࡞࠸㸬ࡍ࡞ࢃࡕ㸪
assay mixture ୰ࡢ⿕㦂≀㉁⃰ᗘࡀ 200 μM ࡼࡾࡶప࠸ሙྜ㸪ROS data ࡀୖグ㜈್ࢆ㉸࠼࡚࠸ࡓሙྜ࡟ࡣ㝧ᛶุᐃࢆ⾜࠺ࡇ࡜ࡀ࡛ࡁࡿࡀ㸪㜈್ࡼࡾࡶప࠸ሙྜ࡟ࡣ㝜ᛶุᐃࡣ࡛ࡁ࡞࠸㸬

3 ヨ㦂ἲࡢࣂࣜࢹ࣮ࢩࣙࣥ
3.1 ⏝࠸ࡽࢀࡓ≀㉁࡜ࡑࡢጇᙜᛶ
ROS ࢔ࢵࢭ࢖ࡢከ᪋タࣂࣜࢹ࣮ࢩ࡛ࣙࣥࡣ 2 ✀㢮ࡢࢯ࣮࣮ࣛࢩ࣑࣮ࣗࣞࢱࡀ౑⏝ࡉࢀ㸪ࡑࢀࡒࢀ 1 ヨ㦂ࡎࡘィ 2 ヨ㦂ࡀᐇ᪋ࡉࢀࡓ㸬ྛヨ㦂ࡣᢏ⾡⛣㌿ᛶࡢホ౯ࢆ┠ⓗ࡜ࡋ࡚ࣉࣞ
ࣂࣜࢹ࣮ࢩࣙࣥ㸦Phase I㸧ヨ㦂࡜ヨ㦂ἲࡢホ౯ࢆ┠ⓗ࡜ࡍࡿࣂࣜࢹ࣮ࢩࣙࣥ㸦Phase II㸧ࡼࡾᵓᡂࡉࢀࡓ㸬ࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ㸪ࣂࣜࢹ࣮ࢩࣙࣥ࡟ඹ㏻ࡋ࡚㸪㝧ᛶᑐ↷≀㉁࡜ࡋ࡚
quinine HCl ࢆ㸪㝜ᛶᑐ↷≀㉁࡜ࡋ࡚ sulisobenzone ࡀ౑⏝ࡉࢀࡓ㸬Quinine HCl ࡣࣄࢺࡢගẘᛶ≀㉁࡜ࡋ࡚ሗ࿌ࡉࢀ࡚࠸ࡿ㸦Ljunggren et al, 1986㸧㸬Sulisobenzone ࡣ 3T3 NRU-PT ࡢࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡛ࣄࢺࢹ࣮ࢱࢆࡶ࡜࡟㠀ගẘᛶ≀㉁࡟ศ㢮ࡉࢀ࡚࠸ࡿ(Spielmann et al,
1998㸧㸬
࡞࠾㸪ࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ࡟⏝࠸ࡓ 5-fluorouracil (5-FU)ࡣࣄࢺ࡛ගẘᛶ≀㉁࡜ࡋ࡚ሗ࿌
ࡉࢀ࡚࠸ࡿ㸦Dillaha et al, 1983㸧ࡀ㸪3T3 NRU-PT ࡛ࡣගẘᛶࢆ♧ࡉ࡞࠸࡜ሗ࿌ࡉࢀ࡚࠸ࡿ

18
24

㸦Kleinman et al, 2010 and Onoue et al, 2010㸧㸬5-FU ࡟ࡼࡿගẘᛶࡢཎᅉ࡜ࡋ࡚㸪UV-B ࡢࡳ࡟཯ᛂࡋ࡚࠸ࡿ࡜ࡢሗ࿌㸦Kirkup M.E. et al, 2003 and Andersen K.E. et al, 1984㸧ࡸ UV-B ࡛ගศゎࡍࡿ㝿ࡢ ROS ⏕ᡂ࡟ࡼࡿ࡜ࡢሗ࿌㸦Miolo G. et al, 2011㸧ࡀ࠶ࡿ㸬5-FU ࡣ⮬↛ࡢኴ㝧ගࡢἼ㛗⠊ᅖ࡛ࡣ୺࡟ UV-B㸦290̽320 nm㸧ࢆ྾཰ࡍࡿ㸦ࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩㸪Appendix 7㸧ࡇ࡜࠿ࡽ㸪5-FU ࡢග໬Ꮫⓗ࡞άᛶ໬࡟ࡣ UV-B ࡢ↷ᑕࡀᚲ㡲࡜⪃࠼ࡽࢀࡿ㸬5-FU ࡢගẘᛶ࡟㛵ࡋ࡚㸪㧗ရ㉁࡞ࣄࢺ࡛ࡢࢹ࣮ࢱࡣ࡞ࡃ㸪5-FU ࡀගẘᛶ≀㉁࡛࠶ࡿ࠿࡝࠺࠿ࡣุ᩿࡛ࡁ
࡞࠿ࡗࡓ㸬5-FU ࡣ㸪3T3 NRU-PT ࡔࡅ࡛࡞ࡃ㸪ROS ࢔ࢵࢭ࢖࡛ࡶ㝧ᛶ⤖ᯝࢆ♧ࡉ࡞࠸࡜ࡢ
᝟ሗࡀ࠶ࡗࡓࡇ࡜࠿ࡽ㸪ROS ࢔ࢵࢭ࢖ࡢ㝈⏺࡟㛵ࡍࡿ᝟ሗࢆ☜ㄆࡍࡿࡓࡵ࡟ࣉࣞࣂࣜࢹ࣮
ࢩࣙࣥ⏝ࡢ໬ྜ≀࡜ࡋ࡚㑅ᢥࡉࢀࡓ㸬୍᪉㸪5-FU ࢆྵࡴࣇࢵ໬ࣆ࣑ࣜࢪࣥ⣔⸆๣ࡢ๪స⏝࡜ࡋ࡚ᡭ㊊⑕ೃ⩌㸦㔜⠜๪స⏝⑌ᝈูᑐᛂ࣐ࢽࣗ࢔ࣝ㸪ᡭ㊊⑕ೃ⩌㸪ཌ⏕ປാ┬㸪2010㸧ࡀ▱ࡽࢀ࡚࠾ࡾ㸪5-FU ࡢගẘᛶ࡜ࡋ࡚␲ࢃࢀ࡚࠸ࡿ㸬ࡋ࠿ࡋ㸪࠸ࢃࡺࡿ໬Ꮫ≀㉁࡟ࡼࡿ┤᥋ⓗ࡞ගẘᛶ࡟ࡼࡿࡶࡢ࡛ࡣ࡞ࡃ㸪ᡭ㊊⑕ೃ⩌ࡢ⑕≧ࡀኴ㝧ග⥺࡬ࡢ᭚㟢࡟ࡼࡗ࡚ᝏ໬ࡋ
ࡓ⑕౛ࡀྵࡲࢀ࡚࠸ࡿྍ⬟ᛶࡶ⪃៖ࡍ࡭ࡁ࠿ࡶࡋࢀ࡞࠸㸬

3.1.1

ࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ

ࣉࣞࣂࣜࢹ࣮ࢩ࡛ࣙࣥࡣ㝜ᛶᑐ↷≀㉁㸪㝧ᛶᑐ↷≀㉁ࢆྵࡴ 13 ≀㉁ࡀ౑⏝ࡉࢀࡓ㸬ࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ࡟౑⏝ࡋࡓ≀㉁࡟㛵ࡍࡿ᝟ሗࢆࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩ࡼࡾᢤ⢋ࡋ㸪Appendix
2 ࠾ࡼࡧ Appendix 3 ࡟♧ࡍ㸬
௚ࡢ 10 ≀㉁ࡢ࠺ࡕ㸪8-MOP㸪amiodarone HCl㸪chlorpromazine㸪doxycycline HCl㸪furosemide㸪
ketoprofen ࠾ࡼࡧ norfloxacin ࡣ 3T3NRU-PT ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡢගẘᛶ≀㉁ࡢࣜࢫࢺ࠿ࡽ
㑅ᢥࡉࢀࡓ㸬ࣄࢺࡢࢹ࣮ࢱࡣ 3T3NRU-PT ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟グ㍕ࡉࢀ࡚࠸ࡿ㸦Spielmann
et al, 1994a and 1998a㸧㸬Diclofenac㸪levofloxacin㸪omeprazole ࡢࣄࢺගẘᛶ≀㉁࡜ࡢሗ࿌ࢆ
᰿ᣐ࡟㑅ᢥࡉࢀࡓ㸦Przybilla et al, 1987㸪Boccumini et al, 2000㸪 Dam et al, 2008㸧㸬

3.1.2

ࣂࣜࢹ࣮ࢩࣙࣥ

ࣂࣜࢹ࣮ࢩࣙࣥ࡟⏝࠸ࡓ໬Ꮫ≀㉁ࡢᵓᡂࢆ⾲ 2 ࡟♧ࡍ㸬ගẘᛶ≀㉁ 23 ≀㉁㸪㠀ගẘᛶ≀㉁ 19 ≀㉁ࡢィ 42 ≀㉁ࢆࢥ࣮ࢻ໬ࡋࡓ≧ែ࡛㸪ヨ㦂᪋タ 7 ᪋タ࡟ศ㓄ࡋࡓ㸬42 ≀㉁ࡢᵓᡂ
ࡣ་⸆ရ 23 ≀㉁㸪໬Ꮫ≀㉁ 9 ≀㉁㸪⣸እ⥺྾཰๣ 10 ≀㉁࡛࠶ࡗࡓ㸬ࣂࣜࢹ࣮ࢩࣙࣥ࡟౑
⏝ࡋࡓ≀㉁ࡢ㑅ᢥ᰿ᣐࢆ Appendix 4 ࡟♧ࡋࡓ㸬

1925

⾲ 2 ࣂࣜࢹ࣮ࢩࣙࣥ࡟⏝࠸ࡽࢀࡓ≀㉁ศ㢮ගẘᛶ≀㉁㸦23 ≀㉁㸧

㠀ගẘᛶ≀㉁㸦19 ≀㉁㸧

་⸆ရ
㸦23 ≀㉁㸧

㸦18 ≀㉁㸧
Acridine
Acridine HCl
Amiodarone HCl
Chlorpromazine HCl
Doxycycline HCl
Fenofibrate
Furosemide
Ketoprofen
6-Methylcoumarine໬Ꮫ≀㉁㸦9 ≀㉁㸧

㸦4 ≀㉁㸧
Anthracene
Bithionol
Hexachlorophene
Rose Bengal

㸦5 ≀㉁㸧
Sodium dodecyl sulphateCinnamic acid
Chlorhexidine
L-histidine
p-Aminobenzoic acid⣸እ⥺྾཰๣
㸦10 ≀㉁㸧

㸦1 ≀㉁㸧
Butyl methoxydibenzoylmethane (Avobenzone)

㸦9 ≀㉁㸧
Benzylindene camphor sulphonic acid
4-Methyl benzylidene camphor
Octyl salicylate
Octyl methoxycinnamate
2-Ethylhexyl 4-methoxycinnamate
2-(2-Hydroxy-5-methylphenyl)benzotriazole
2-tert-Butyl-6-(5-chloro-2H-benzotriazol-2-yl)-4-methylphenol
2-(2H-Benzotriazol-2-yl)-4-(1,1,3,3-tetramet
hylbutyl)phenol
2-(2H-Benzotriazol-2-yl)-6-dodecyl-4-methylphenol

8-Methoxy psoralen
Nalidixic acid
Nalidixic acid (Nasalt)
Norfloxacin
Ofloxacin
Piroxicam
Promethazine HCl
Rosiglitazone
Tetracycline㸦5 ≀㉁㸧
AspirinBenzocaine
Erythromycin
Phenytoin
Penicilin G

3.1.3 ヨ㦂ἲࡢṇ☜ᛶࢆホ౯ࡍࡿࡓࡵ࡟⏝࠸ࡽࢀࡓ໬Ꮫ≀㉁ࡢ invivo ཧ↷ࢹ࣮ࢱ
ROS ࢔ࢵࢭ࢖ࡢከ᪋タࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡛ࡣ㸪ࢥ࣮ࢻ໬ࡋࡓ໬ྜ≀ࢆ⏝࠸࡚ᐇ᪋ࡋࡓ
ࣉࣞࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡢ⤖ᯝࢆ⏝࠸࡚ヨ㦂ἲࡢṇ☜ᛶࢆホ౯ࡋࡓ㸬ࣂࣜࢹ࣮ࢩࣙࣥ࡟⏝࠸ࡓ 42 ≀㉁ࡢග໬Ꮫⓗ≉ᛶ࠾ࡼࡧࣄࢺ㸪ື≀㸪in vitro ホ౯⣔࡛ࡢගẘᛶ࡟㛵ࡍࡿ᝟ሗࢆࣂ
ࣜࢹ࣮ࢩࣙࣥሗ࿌᭩ࡼࡾᢤ⢋ࡋ㸪Appendix 5 ࡟♧ࡋࡓ㸬౑⏝ࡋࡓ≀㉁ࡢ࠺ࡕ㸪ගẘᛶ≀㉁࡜
㠀ගẘᛶ≀㉁ࡀ࡯ࡰ༙ࠎ࡛࠶ࡿࡇ࡜ࡣឤᗘࡸ≉␗ᗘ➼㸪ヨ㦂ἲࡢṇ☜ᛶࢆホ౯ࡍࡿࡓࡵ࡟
ᐇ᪋ࡍࡿࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟㐺ࡋ࡚࠸ࡓ࡜ᛮࢃࢀࡿ㸬ࡋ࠿ࡋ࡞ࡀࡽ㸪་⸆ရ࡛ࡣගẘᛶ≀㉁ࡢ๭ྜࡀከࡃ㸪⣸እ⥺྾཰๣࡛ࡣ㠀ගẘᛶ≀㉁ࡢ๭ྜࡀከࡃ㸪≀㉁ࡢ✀㢮࡟ࡼࡗ࡚㸪㑅ᐃࡉࢀࡓ㝧ᛶ≀㉁࡜㝜ᛶ≀㉁࡟೫ࡾࡀ࠶ࡗࡓ㸬

20
26

࡞࠾㸪➨୕⪅ホ౯ጤဨ఍ࡣࡑࡢሗ࿌᭩࡟࠾࠸࡚㸪౑⏝ࡋࡓ໬Ꮫ≀㉁࡟㛵ࡋࠕ➨୕⪅ホ౯ጤဨ఍ࡣࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡛㑅ᢥࡉࢀࡓ໬Ꮫ≀㉁ࡣ࢔ࢵࢭ࢖ࢆホ౯ࡍࡿ໬Ꮫ≀㉁ࡢ௦⾲࡜ࡋ࡚㐺ษ࠿ࡘ༑ศ࡞࡛࠶ࡿࡇ࡜࡟ྠពࡋࡓ㸬ࣄࢺගẘᛶ≀㉁ࢆྵࡴ 42 ≀㉁ࡣ㝧ᛶ࡜ࡋ࡚▱ࡽࢀࡿ 23 ≀㉁㸪㝜ᛶ 19 ≀㉁ࡼࡾᵓᡂࡉࢀࡓ㸬ࡑࢀࡽࡢ≀㉁ࡣࣄࢺࣃࢵࢳࢸࢫࢺ࡜ 3T3
NRU-PT ࡢࢹ࣮ࢱ࡟⿬௜ࡅࡽࢀ࡚࠸ࡓ㸬ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡛ᚓࡽࢀࡓࡍ࡭࡚ࡢࢹ࣮ࢱࡣࣂ
ࣜࢹ࣮ࢩࣙࣥヨ㦂ሗ࿌᭩࡟グ㍕ࡉࢀ࡚࠸ࡿ㸬ࠖ࡜グ㍕ࡋ࡚࠸ࡿ㸬

3.2 ࣂࣜࢹ࣮ࢩࣙࣥ⤖ᯝࡢホ౯
3.2.1 ヨ㦂ἲࡢࢹ࣮ࢱ࡜⤖ᯝࡢ฼⏝ᛶ
ROS ࢔ࢵࢭ࢖ከ᪋タࣂࣜࢹ࣮ࢩ࡛ࣙࣥࡣ 2 ✀㢮ࡢࢯ࣮࣮ࣛࢩ࣑࣮ࣗࣞࢱࡀ౑⏝ࡉࢀ㸪2
ヨ㦂ᐇ᪋ࡉࢀࡓ㸬1 ヨ㦂┠ࡣ࢔ࢺࣛࢫ♫ࡢ Suntest CPS/CPS+ࢆ⏝࠸㸪2011 ᖺ 3 ᭶㹼10 ᭶࡟࠿ࡅ࡚㸪3 ᪋タ࡛ᐇ᪋ࡉࢀ㸪ࡶ࠺ 1 ヨ㦂ࡣࢭࣜࢵࢡ♫ࡢ SXL-2500V2 ࢆ⏝࠸㸪2011 ᖺ 5 ᭶㹼2012 ᖺ 6 ᭶࡟࠿ࡅ࡚ 4 ᪋タ࡛ᐇ᪋ࡉࢀࡓ㸬ࡇࢀࡽࡢࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡢࢹ࣮ࢱ࡜⤖ᯝࡣࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩୰࡟グ㍕ࡉࢀ㸪฼⏝ྍ⬟࡛࠶ࡿ㸬

3.2.2 ヨ㦂᪉ἲࡢṇ☜ᛶࣂࣜࢹ࣮ࢩࣙࣥヨ㦂⤖ᯝ࠿ࡽ⟬ฟࡋࡓヨ㦂᪋タࡢឤᗘ㸪≉␗ᗘ㸪㝧ᛶண⋡㸪㝜ᛶண⋡࠾ࡼࡧṇ☜ᛶࢆ⾲ 3 ࡟♧ࡋࡓ㸬㞴⁐ᛶࡢࡓࡵホ౯୙ྍ⬟࡛࠶ࡗࡓ≀㉁ࢆ㝖ࡃ࡜㸪ගẘᛶ≀㉁࡟ᑐࡍࡿឤᗘࡣ඲᪋タ࡜ࡶ࡟ 100%࡛࠶ࡾ㸪㝜ᛶண⋡ࡶྛ᪋タ࡜ࡶ 100%࡛࠶ࡾ㸪ഇ㝜ᛶࡣ࡞࠿ࡗࡓ㸬21
27

⾲ 3 ROS ࢔ࢵࢭ࢖ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂⤖ᯝ
ヨ㦂
᪋タ

ឤᗘ

≉␗ᗘ

㝧ᛶ
ண ⋡

㝜ᛶ
ண ⋡

ṇ☜ᛶ

100%
(21/21)75.0%(12/16)84.0%(21/25)100%(12/12)

89.2%(33/37)100%(18/18)71.4%(10/14)

81.8%(18/22)100%(10/10)87.5%(28/32)

3

100%(20/20)88.2%(15/17)90.9%(20/22)100%(15/15)

94.6%(35/37)4

100%(19/19)71.4%(10/14)

82.6%(19/23)100%(10/10)87.9%(29/33)5

100%(19/19)50.0%(6/12)

76.0%(19/25)

100%
(6/6)

80.6%
(25/31)100%(19/19)73.3%(11/15)

82.6%(19/23)100%(11/11)

88.2%(30/34)100%(18/18)69.2%(9/13)

81.8%(18/22)

100%
(9/9)

87.1%
(27/31)ேᕤኴ㝧ⅉග※

1
2

6
7

࢔ࢺࣛࢫ♫
SuntestCPS/CPS+ࢭࣜࢵࢡ♫SXL-2500V2

Weakly photoreactive ࡜ุᐃࡉࢀࡓ≀㉁ࢆගẘᛶ≀㉁࡜ุᐃࡋࡓሙྜࡢุᐃ⤖ᯝࢆ♧ࡍ㸬ឤᗘ㸸ගẘᛶ≀㉁ࡀ ROS ࢔ࢵࢭ࢖࡛㝧ᛶࢆ♧ࡍ๭ྜ㸦㸣㸧≉␗ᗘ㸸㠀ගẘᛶ≀㉁ࡀ ROS ࢔ࢵࢭ࢖࡛㝜ᛶࢆ♧ࡍ๭ྜ㸦㸣㸧㝧ᛶண ⋡㸸ROS ࢔ࢵࢭ࢖࡛㝧ᛶࡔࡗࡓ≀㉁ࡢ࠺ࡕ㸪ගẘᛶ≀㉁ࡢ๭ྜ㸦㸣㸧㝜ᛶண ⋡㸸ROS ࢔ࢵࢭ࢖࡛㝜ᛶࡔࡗࡓ≀㉁ࡢ࠺ࡕ㸪㠀ගẘᛶ≀㉁ࡢ๭ྜ㸦㸣㸧ṇ☜ᛶ㸸ROS ࢔ࢵࢭ࢖ࡢุᐃ⤖ᯝࡀගẘᛶ≀㉁㸪㠀ගẘᛶ≀㉁ࢆṇࡋࡃุᐃࡋࡓ๭ྜ㸦㸣㸧

3.2.3 ヨ㦂ࢹ࣮ࢱࡢ෌⌧ᛶྛ᪋タ࡟࠾ࡅࡿ㝧ᛶᑐ↷≀㉁㸪㝜ᛶᑐ↷≀㉁ࡢ᪥ෆኚືࢆᅗ 5 ࡟㸪᪥㛫ኚືࢆᅗ 6 ࡟♧ࡋ㸪ࡲࡓ㸪ග※⿦⨨㛫ࡢ෌⌧ᛶࢆᅗ 7 ࡟♧ࡋࡓ㸬᪥ෆኚື㸪᪥㛫ኚື࡜ࡶ࡟ᑠࡉࡃ㸪᪋タ㛫㸪␗࡞ࡿග※⿦⨨㛫ࡢኚືࡶᑠࡉ࠿ࡗࡓ㸬ヨ㦂⣔ࡢ᪋タෆ㸪᪋タ㛫ࡢ෌⌧ᛶࡣ㧗ࡃ㸪ග※⿦⨨㛫ࡢỗ⏝ᛶ࡟ၥ㢟࡞࠿ࡗࡓ㸬ࡲࡓ㸪ྛ᪋タ࡟࠾ࡅࡿヨ㦂⤖ᯝࡢ෌⌧ᛶ࡟ࡘ࠸࡚㸪⾲ 4
࡟♧ࡋࡓ㸬

22
28

㻌㻌

㻌

ᅗ5

ྛヨ㦂᪋タ࡟࠾ࡅࡿ㝧ᛶᑐ↷≀㉁㸪㝜ᛶᑐ↷≀㉁ࡢ᪥ෆኚື
᪥ෆኚືࡣྛ᪋タ࡛᭱ࡶከࡃࡢ ᐃࢆᐇ᪋ࡋࡓ᪥ࡢ㝧ᛶ࠾ࡼࡧ㝜ᛶᑐ↷≀㉁ࡢ⤖ᯝ࠿ࡽ⟬ฟࡋࡓ㸬Lab#1–3: Atlas Suntest CPS(⣙ 2.0 mW/cm2)㸪Lab#4–7: Seric SXL-2500V2 (3.0–5.0 mW/cm2).,‫ۍ‬: quinine ࡢ singlet oxygen, ‫ی‬: quinine ࡢ superoxide; ‫ۑ‬: sulisobenzone ࡢ singlet oxygen, ‫ە‬:
sulisobenzone ࡢ superoxide, ࢹ࣮ࢱࡣ meansSD ࢆ♧ࡍ(n=9)㸬

ᅗ6

ྛヨ㦂᪋タ࡟࠾ࡅࡿ㝧ᛶᑐ↷≀㉁㸪㝜ᛶᑐ↷≀㉁ࡢ᪥㛫ኚື᪥㛫ኚືࡣྛ᪋タࡢ ᐃ᪥࡟࠾ࡅࡿ᭱ึࡢ ᐃ࡟࠾ࡅࡿ㝧ᛶ࠾ࡼࡧ㝜ᛶᑐ↷≀㉁ࡢ⤖ᯝ࠿ࡽ⟬ฟࡋࡓ㸬Lab#1–3: Atlas Suntest CPS(⣙ 2.0 mW/cm2)㸪Lab#4–7: Seric SXL-2500V2 (3.0–5.0 mW/cm2).,‫ۍ‬: quinine ࡢ singlet oxygen, ‫ی‬: quinine ࡢ superoxide; ‫ۑ‬: sulisobenzone ࡢ singlet oxygen, ‫ە‬:
sulisobenzone ࡢ superoxide, ࢹ࣮ࢱࡣ meansSD ࢆ♧ࡍ(days 1, 2, and 3; n=9)㸬

㻌

23
29

㻌
ᅗ7

㝧ᛶᑐ↷≀㉁㸪㝜ᛶᑐ↷≀㉁ࡢග※⿦⨨㛫ࡢ෌⌧ᛶ᪥㛫ኚືࡣྛ᪋タࡢ ᐃ᪥࡟࠾ࡅࡿ᭱ึࡢ ᐃ࡟࠾ࡅࡿ㝧ᛶ࠾ࡼࡧ㝜ᛶᑐ↷≀㉁ࡢ⤖ᯝ࠿ࡽ⟬ฟࡋࡓ㸬Lab#1̽3: Atlas Suntest CPS(⣙ 2.0 mW/cm2)㸪Lab#4̽7: Seric SXL-2500V2 (3.0̽5.0 mW/cm2).,‫ۍ‬: quinine ࡢ singlet oxygen, ‫ی‬: quinine ࡢ superoxide; ‫ۑ‬: sulisobenzone ࡢ singlet oxygen, ‫ە‬:
sulisobenzone ࡢ superoxide, ࢹ࣮ࢱࡣ meansSD ࢆ♧ࡍ(days 1, 2, and 3; n=9)㸬

⾲ 4 ࣂࣜࢹ࣮ࢩࣙࣥ⤖ᯝࡢග※⿦⨨㛫㸪᪋タ㛫ࡢ෌⌧ᛶࣂࣜࢹ࣮ࢩࣙࣥ࡟⏝࠸ࡓ໬Ꮫ≀㉁
ගẘᛶ≀㉁

㝧ᛶ
ROS

㠀ගẘᛶ≀㉁Suntest CPS

SXL-2500V2

Suntest CPS

SXL-2500V2[#1] 21[#4] 19[#1] 4[#4] 4[#2] 18[#5] 19[#2] 4[#5] 6[#3] 20[#6] 19[#3] 2[#6] 4[#7] 18

࢔ࢵࢭ࢖[#7] 4ࣂࣜࢹ࣮ࢩࣙ

Suntest CPS

SXL-2500V2

Suntest CPS

SXL-2500V2ࣥ⤖ᯝ[#1] 0[#4] 0[#1] 12[#4] 10[#2] 0[#5] 0[#2] 10[#5] 6[#3] 0[#6] 0[#3] 15[#6] 11㝜ᛶ

[#7] 0[#7] 9

Lab#1̽3: Atlas Suntest CPS(⣙ 2.0 mW/cm2)㸪Lab#4̽7: Seric SXL-2500V2 (3.0̽5.0 mW/cm2)
ᩘ್ࡣ ROS ࢔ࢵࢭ࢖࡛㝧ᛶ࠶ࡿ࠸ࡣ㝜ᛶࢆ♧ࡋࡓࢆ♧ࡋࡓ≀㉁ࡢ࠺ࡕ㸪ගẘᛶ≀㉁࠶ࡿ࠸ࡣ㠀ගẘᛶ≀㉁ࡢಶᩘࢆ♧ࡍ㸬

2430

3.2.4 ヨ㦂᪉ἲࡢࢹ࣮ࢱࡢಙ㢗ᛶࣂࣜࢹ࣮ࢩࣙࣥ࡟ཧຍࡋࡓࡍ࡭࡚ࡢヨ㦂᪋タࡀ᪥ᖖⓗ࡟ GLP 㐺ྜヨ㦂ࢆᐇ᪋ࡋ࡚࠸ࡿ᪋タ࡛ࡣ࡞࠸ࡀ㸪ࢹ࣮ࢱࡢಙ㢗ᛶಖドࡣ࡞ࡉࢀࡓ㸬ࡍ࡞ࢃࡕ㸪ࡍ࡭࡚ࡢ⏕ࢹ࣮ࢱ࠾ࡼࡧゎᯒ⤖ᯝࡣྛ᪋タ࡟࠾࠸࡚☜ㄆࡉࢀ㸪ࡑࡢᚋ㸪ࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀဨ఍ࡢಙ㢗ᛶಖドࢢ࣮ࣝࣉ࡛☜ㄆࡉࢀ࡚࠸ࡓ㸬ࡇࢀࡽࡢ᧯సࢆ㏻ࡌ㸪ಙ㢗ᛶಖドࡢほⅬ࡛㸪⏕ࢹ࣮ࢱࡀṇ☜࡟ヨ㦂⤖ᯝ࡟཯ᫎࡉࢀ࡚࠸ࡿࡇ࡜ࡀ☜ㄆࡉࢀ࡚࠸ࡓ㸬

4 ヨ㦂᪉ἲ࡟㛵ࡍࡿ௚ࡢ⛉Ꮫⓗ⪃ᐹ
4.1 ග࡟୙Ᏻᐃ࡞≀㉁࡬ࡢ㐺⏝
ROS ࢔ࢵࢭ࢖ࡣ་⸆ရࡢග໬Ꮫⓗ཯ᛂᛶࢆ㎿㏿࠿ࡘ⡆౽࡟ᐃ࡛ࡁࡿࡀ㸪ࡑࡢ཯㠃㸪ᚓࡽࢀࡿࢹ࣮ࢱ࠿ࡽࡣගᏳ඲ᛶࡔࡅ࡛ࡣ࡞ࡃ㸪ගᏳᐃᛶ࡞࡝࡟ၥ㢟ࡀ࠶ࡿ໬ྜ≀ࡶྵࡵ࡚㝧ᛶ࡜ุูࡍࡿࡇ࡜ࡀ࠶ࡿ㸬ࡇࢀࡣ ROS ࢔ࢵࢭ࢖ࡀගẘᛶ཯ᛂࡢࡁࢃࡵ࡚ึᮇ࡟㉳ࡇࡿග໬Ꮫⓗ཯ᛂᛶࢆホ౯ࡍࡿࢶ࣮࡛ࣝ࠶ࡿࡀᨾࡢㄢ㢟࡛࠶ࡾ㸪ගᏳ඲ᛶホ౯࡟࠾ࡅࡿ୍ḟࢫࢡ࣮ࣜࢽࣥࢢ࡜ࡋ࡚ᮏἲࢆ฼⏝ࡍࡿ㝿࡟ࡣࡇࡢⅬࢆ⇍⪃ࡢୖࢹ࣮ࢱࡢゎ㔘ࢆ⾜࠺ᚲせࡀ࠶ࡿ㸬Onoue ࡽࡣࡇࡢㄢ㢟ࢆゎỴࡍ࡭ࡃ㸪ヨ㦂⁐ᾮ࡜ඹ࡟ᣦ♧⸆ࢆῧຍࡋ࡞࠸ヨ㦂ࢥࣥࢺ࣮ࣟࣝ⁐ᾮࢆㄪ〇ࡋ㸪཯ᛂᚋ࡟཯ᛂ⁐ᾮ࠿ࡽヨ㦂ࢥࣥࢺ࣮ࣟࣝ⁐ᾮ࡛ᚓࡽࢀࡓ್ࢆᕪࡋᘬࡃᡭἲࢆ
ᥦ᱌ࡋ࡚࠾ࡾ㸪ගᏳᐃᛶ࡟ၥ㢟ࡢ࠶ࡿ໬ྜ≀࡟ᑐࡍࡿண

⢭ᗘࡢᨵၿࡀㄆࡵࡽࢀ࡚࠸ࡿ

(Onoue et al., 2013)㸬

4.2 㞴⁐ᛶ≀㉁࡬ࡢᑐᛂ
㏆ᖺࡢ๰⸆άື࡟࠾࠸࡚ࡣỈ࡟㞴⁐ᛶࡢ໬ྜ≀ࡀ་⸆ရೃ⿵≀㉁࡜ࡋ࡚๰ฟࡉࢀࡿࡇ࡜
ࡀ㠀ᖖ࡟ከ࠸ࡀ㸪ROS ࢔ࢵࢭ࢖࡟࠾࠸࡚ࡣᇶᮏⓗ࡟ 200 μM ࡢ⃰ᗘ࡛ࢫࢡ࣮ࣜࢽࣥࢢࢆ⾜࠺ࡓࡵ࡟ assay mixture ୰࡛ࡢᯒฟࢆㄆࡵࡿࡇ࡜ࡀ࠶ࡿ㸬ࡑࡢሙྜ࡟ࡣࡼࡾప⃰ᗘ࡟࡞ࡿࡼ
࠺ᕼ㔘ࢆ⾜ࡗ࡚ ROS ࢔ࢵࢭ࢖ࢆᐇ᪋ࡍࡿࡇ࡜ࡀྍ⬟࡛ࡣ࠶ࡿࡀ㸪ࡑࡢ㝿࡟ࡣᚓࡽࢀࡓࢹ࣮
ࢱ࡟ࡼࡗ࡚㝧ᛶุᐃࢆฟࡍࡇ࡜ࡣ࡛ࡁ࡚ࡶ㝜ᛶุᐃࢆฟࡍࡇ࡜ࡀࡁࢃࡵ࡚㞴ࡋ࠸㸬Onoueࡽࡣࡇࡢㄢ㢟ࢆゎỴࡍ࡭ࡃ㸪⏺㠃άᛶ๣ࢆ฼⏝ࡋࡓ ROS ࢔ࢵࢭ࢖ࡸ࢔ࣝࣈ࣑ࣥࢆ assay
mixture ࡟ຍ࠼ࡓᨵⰋᆺ ROS ࢔ࢵࢭ࢖ࢆᥦ᱌ࡋ࡚࠾ࡾ㸪ᮏᡭἲ࡛ࡣ㞴⁐ᛶ་⸆ရ࡬ࡢ㐺⏝⋡ࡀ㢧ⴭ࡟ᨵၿࡉࢀࡿ (Onoue et al., 2008; Onoue et al., 2014; Seto et al., 2013)㸬⏺㠃άᛶ๣ࢆ฼⏝ࡋࡓᨵⰋᆺ ROS ࢔ࢵࢭ࢖࡟ࡘ࠸࡚ࡣ㸪㞴Ỉ⁐ᛶࡢ໬⢝ရ⣲ᮦ࡟࠾࠸࡚ࡶࡑࡢ᭷⏝ᛶࡀ☜ㄆࡉࢀ࡚࠾ࡾ㸪ROS ࢔ࢵࢭ࢖ࡢ㐺⏝ᛶᣑ኱࡟኱ࡁࡃᐤ୚ࡍࡿࡶࡢ࡜ᮇᚅࡉࢀࡿ㸦Onoue etal., 2013㸧㸬ࡓࡔࡋ㸪assay mixture ࡢ⤌ᡂࢆኚ࠼ࡓሙྜ࡟ࡣ ROS ࢔ࢵࢭ࢖࡟࠾ࡅࡿุᐃᇶ

25
31‽ࡀኚࢃࡿྍ⬟ᛶࡀ࠶ࡾ㸪ㄗࡗࡓගᏳ඲ᛶ᝟ሗࢆᚓࡿࡇ࡜ࡀ࡞࠸ࡼ࠺⣽ᚰࡢὀពࡀᚲせ࡛࠶ࡿ㸬

4.3 ΰྜ≀࠾ࡼࡧศᏊ㔞୙᫂ヨᩱ࡬ࡢᑐᛂ
໬⢝ရ⣲ᮦ࡟࠾࠸࡚ࡣᐃࡲࡗࡓศᏊ㔞ࢆ᭷ࡋ࡚࠸࡞࠸᳜≀ᢳฟ≀➼ࡢΰྜ≀ࡀᩘከࡃᏑ
ᅾࡍࡿࡀ㸪ROS ࢔ࢵࢭ࢖࡟࠾࠸࡚ࡣ㸪๓㏙ࡢ㏻ࡾ 200 ȝM ࡢ⃰ᗘ࡛ࢫࢡ࣮ࣜࢽࣥࢢࢆ⾜࠺ࡓࡵ࡟㸪ࣔࣝ⃰ᗘࡢ⟬ฟ࡛ࡁ࡞࠸ΰྜ≀ࡢヨ㦂ᐇ᪋ࡀ୙ྍ⬟࡛࠶ࡿ㸬Nishida ࡽࡣࡇࡢㄢ㢟ࢆゎỴࡍ࡭ࡃ㸪ヨ㦂⃰ᗘࢆศᏊ㔞ࡀ᫂☜࡛࡞࠸࡜タᐃ࡛ࡁ࡞࠸ࣔࣝ⃰ᗘ࡛ࡣ࡞ࡃ㸪μg/mL༢఩࡛ࡢ ROS ࢔ࢵࢭ࢖ࢆሗ࿌ࡋ࡚࠸ࡿ (Nishida et al., 2015)㸬ᮏሗ࿌࡟౑⏝ࡉࢀࡓ≀㉁ࢆ⾲
5 ࡟♧ࡋ㸪ࡲࡓ㸪ᚓࡽࢀࡓ⤖ᯝࢆ⾲ 6 ࡟♧ࡋࡓ㸬ሗ࿌࡛ࡣ㸪ࡇࢀࡲ࡛࡟ගᏳ඲ᛶୖࡢᠱᛕࡀ
Ꮡᅾࡍࡿ࡜ሗ࿌ࡉࢀ࡚࠸ࡿ໬Ꮫ≀㉁ࡢ࡯࡜ࢇ࡝ࡀศᏊ㔞 500 ௨ୗࡢపศᏊ໬ྜ≀࡛࠶ࡾ㸪ࡑࡢᖹᆒศᏊ㔞ࡣ 250 ࡜௬ᐃ࡛ࡁࡿࡇ࡜㸪ศᏊ㔞 250 ࡢ໬ྜ≀ࡀ 200 ȝM ࡜࡞ࡿ 50 ȝg/mL࡛ࡢヨ㦂࡛㸪ᐃἲࡢ ROS ࢔ࢵࢭ࢖࡜ྠ➼ࡢឤᗘࡀㄆࡵࡽࢀࡿࡇ࡜ࡀ☜ㄆࡉࢀ࡚࠸ࡿ㸬ΰྜ≀ࢆ⏝࠸ࡓ᳨ド࡟࠾࠸࡚ࡶ㸪㐺⏝⃰ᗘ 50 ȝg/mL ࡛ࡢヨ㦂ࡣ㝧ᛶ᳨ฟ⋡ 100%࡛࠶ࡿ᭷⏝࡞ࢫࢡ࣮ࣜࢽࣥࢢࢶ࣮࡛ࣝ࠶ࡿࡇ࡜ࡀ☜ㄆࡉࢀࡓࡇ࡜࠿ࡽ㸪ࣔࣝ⃰ᗘࡢタᐃ࡛ࡁ࡞࠸⣲ᮦ࡬ࡢ ROS ࢔ࢵࢭ࢖ᒎ㛤ࡢྍ⬟ᛶࡀ♧ࡉࢀࡓ㸬ࡓࡔࡋΰྜ≀࡟ࡼࡗ࡚㸪᳜≀ᢳฟ≀ࡢࡼ࠺࡟㸪ࡑࡢෆᐜ≀ࡀ୙᫂░࡛࠶ࡿሙྜࡀ⪃࠼ࡽࢀ㸪ྠࡌྡ๓ࡢ⣲ᮦ࡟࠾࠸࡚ࡶࡑࡢᵓᡂᡂศࡸ⤌ᡂࡀ኱ࡁࡃ␗࡞ࡿྍ⬟ᛶࡀ࠶ࡿ㸬ࡇࡢࡼ࠺࡞ሙྜ࡟࠾࠸࡚ࡣ㸪ROS ࢔ࢵࢭ࢖࡛ᚓࡽࢀࡿ⤖ᯝ࡟ࡶ኱ࡁ࡞ᙳ㡪ࢆཬࡰࡍࡇ࡜ࡀ⪃࠼ࡽࢀࡿࡓࡵ㸪ㄗࡗࡓගᏳ඲ᛶ᝟ሗࢆᚓࡿࡇ࡜ࡀ↓࠸ࡼ࠺࡟⣽ᚰࡢὀពࡀᚲせ࡛࠶ࡿ㸬26
32

⾲ 5 ヨ㦂࡟⏝࠸ࡽࢀࡓ≀㉁ศ㢮ගẘᛶ≀㉁㸦40 ≀㉁㸧

໬⢝ရ㸦30 ≀㉁㸧

㸦17 ≀㉁㸧
4-Methyl-7-ethoxyco
umarin5-Methoxypsoralen
6-Methylcoumarin
7-Methoxycoumarin
8-Methoxypsoralen
Benzophenone
Bithionol
Dichlorophene
Fenticlorࡑࡢ௚≀㉁㸦16 ≀㉁㸧

㸦12 ≀㉁㸧
Acridine
Amiodarone HCl
Chlorpromazine HCl
Diclofenac Na
Enoxacin
Fenofibrateΰྜ≀㸦20 ≀㉁㸧

㸦11 ≀㉁㸧
Angelica root oil
Bergamot oil
Celery seed oil
Cumin seed oil
Grapefruit oil
Lemon oilLime oilOil parsley
St. John’s wort powder
Tagetes oil
Verbena oil⾲ 6㠀ගẘᛶ≀㉁㸦26 ≀㉁㸧
Hexachlorophene
Methyl ȕ-naphthyl
ketoneMethyl N-methyl
anthranilate
Muk ambretteMusk ketone
Musk xylene
p-Phenylenediamine
Triclocarban

Indomethacin
Ketoprofen
Piroxicam
Promethazine HCl
Sulfanilamide
Tetracycline HCl㸦13 ≀㉁㸧
1,3-Butylene glycol
2-Propanol
3-(-4-Methylbenzylidene) camphor
Ascorbic acid
Cetyl alcohol
Ethanol
Glycerine
Isopropyl myristate
Lauric acid
Propylene glycol
Sodium laurate
Sodium lauryl sulfate
Sulisobenzone
㸦4 ≀㉁㸧DMSO
Lactic acid
Methyl salicylate
Penicillin G㸦9 ≀㉁㸧
Carrot oil
Corn oilGinger oil
Lemongrass oil
Olive oilOrange oil
Rape seed oil
Safflower oil
Soybean oil

ROS ࢔ࢵࢭ࢖ヨ㦂⤖ᯝ (50 ȝg/mL)

ศ㢮

ឤᗘ

≉␗ᗘ

㝧ᛶ
ண ⋡

㝜ᛶ
ண ⋡

ṇ☜ᛶ

໬⢝ရ

100%
(17/17)76.9%(10/13)85.0%(17/20)100%(10/10)90.0%(27/30)

ࡑࡢ௚
≀㉁

100%
(12/12)

50.0%
(2/4)

85.7%
(12/14)

100%
(2/2)

87.5%
(14/16)ΰྜ≀

100%(11/11)

55.6%
(5/9)

73.3%
(11/15)

100%
(5/5)

80.0%
(16/20)50 ȝg/mL ࡛ヨ㦂ࢆᐇ᪋ࡋ㸪ุᐃᇶ‽ࡣᚑ᮶ἲ࡟ᚑࡗࡓሙྜࡢ⤖ᯝࢆ♧ࡍ㸬ឤᗘ㸸ගẘᛶ≀㉁ࡀ ROS ࢔ࢵࢭ࢖࡛㝧ᛶࢆ♧ࡍ๭ྜ㸦㸣㸧≉␗ᗘ㸸㠀ගẘᛶ≀㉁ࡀ ROS ࢔ࢵࢭ࢖࡛㝜ᛶࢆ♧ࡍ๭ྜ㸦㸣㸧㝧ᛶண ⋡㸸ROS ࢔ࢵࢭ࢖࡛㝧ᛶࡔࡗࡓ≀㉁ࡢ࠺ࡕ㸪ගẘᛶ≀㉁ࡢ๭ྜ㸦㸣㸧㝜ᛶண ⋡㸸ROS ࢔ࢵࢭ࢖࡛㝜ᛶࡔࡗࡓ≀㉁ࡢ࠺ࡕ㸪㠀ගẘᛶ≀㉁ࡢ๭ྜ㸦㸣㸧ṇ☜ᛶ㸸ROS ࢔ࢵࢭ࢖ࡢุᐃ⤖ᯝࡀගẘᛶ≀㉁㸪㠀ගẘᛶ≀㉁ࢆṇࡋࡃุᐃࡋࡓ๭ྜ㸦㸣㸧27
33

4.4 ග࢔ࣞࣝࢠ࣮ᛶண࡬ࡢ฼⏝໬⢝ရ⣲ᮦ࡟࠾࠸࡚㸪ගᏳ඲ᛶࡣගẘᛶ࡜ග࢔ࣞࣝࢠ࣮ᛶ࡟ศࡅࡽࢀࡿࡀ㸪ࡑࡢ࠺ࡕ㸪ග࢔ࣞࣝࢠ࣮ᛶࡣ㸪ගࢆ௓ࡋࡓ≀㉁࡜ࢱࣥࣃࢡ㉁ࡢ┤᥋⤖ྜࡲࡓࡣ㸪ග࡟ࡼࡿศゎ≀࡜ࢱࣥࣃࢡ㉁ࡢ⤖ྜ࡟ࡼࡿᢠཎᙧᡂࡀ㔜せ࡛࠶ࡿ㸬ග↷ᑕ࡟ࡼࡿບ㉳ගẘᛶ≀㉁⏘⏕᫬࡟Ⓨ⏕ࡍࡿ singlet oxygen ࡜ superoxide ࡣ㸪ග่⃭ᛶࢆច㉳ࡍࡿẘᛶບ㉳≀㉁ࡔࡅ࡛ࡣ࡞ࡃ㸪ග࢔ࣞࣝࢠ࣮ᛶࢆ♧ࡍ≀㉁࡛ࡶྠᵝ࡟Ⓨ⏕ࡋ㸪ග࢔ࣞࣝࢠ࣮ᛶ࡛ࡣ㸪ගࢱࣥࣃࢡ⤖ྜࡲࡓࡣග
ศゎࡢ࢚ࢿࣝࢠ࣮࡟ࡘ࡞ࡀࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿ (Tokura, 2009)㸬୍⯡࡟㸪ග࢔ࣞࣝࢠ࣮ᛶࡢ
ᐃ⩏ࡀᐃࡲࡗ࡚࠾ࡽࡎ㸪⮫ᗋࡢ⌧ሙ࡟࠾࠸࡚ࡶගẘᛶ࡜ග࢔ࣞࣝࢠ࣮ᛶࡀ᫂☜࡟ศࡅࡽࢀ
࡚࠸࡞࠸ሙྜࡀከ࠸࡜⪃࠼ࡽࢀࡿ㸬ࡇࡢࡼ࠺࡞⌧≧࡟࠾࠸࡚ࡣ㸪ගẘᛶࢆ♧ࡉ࡞࠸ග࢔ࣞ
ࣝࢠ࣮ᛶ≀㉁ࡢ㑅ᐃࡀ㞴ࡋ࠸ࡓࡵ㸪ROS ࢔ࢵࢭ࢖ࡀస⏝ᶵᗎࡢほⅬ࠿ࡽග࢔ࣞࣝࢠ࣮ᛶ≀㉁ࡢ᳨ฟࡀྍ⬟࠿ྰ࠿ࢆ᳨ドࡍࡿࡇ࡜ࡣᐜ࡛᫆ࡣ࡞࠸㸬ࡑࡇ࡛ Onoue ࡽࡣ㸪ග࢔ࣞࣝࢠ࣮
ᛶ≀㉁࡜ࡋ࡚ሗ࿌ࡉࢀ࡚࠸ࡿ≀㉁ࡢ୰࡛㸪In vivo ගẘᛶヨ㦂࡟࠾࠸࡚㝜ᛶ⤖ᯝࡀሗ࿌ࡉࢀ
࡚࠸ࡿ≀㉁㸪ࡶࡋࡃࡣ 3T3 NRU-PT ࢆᐇ᪋ࡋ㸪㝜ᛶࡶࡋࡃࡣ␲㝧ᛶࢆ♧ࡋࡓ≀㉁ࢆග࢔ࣞࣝ
ࢠ࣮ᛶࡢᐤ୚ࡀ㧗࠸≀㉁࡜ࡋ࡚ศ㢮ࡋࡓ㸬ග࢔ࣞࣝࢠ࣮ᛶ≀㉁࡜ࡋ࡚ศ㢮ࡉࢀࡓ 18 ≀㉁ࢆ
⾲ 7 ࡟♧ࡋࡓ㸬ࡑࢀࡽ඲࡚ࡢ ROS ࢔ࢵࢭ࢖ࡢ⤖ᯝࡣ㝧ᛶ࡛࠶ࡗࡓ㸬ᚑࡗ࡚㸪ගẘᛶ࡟ග࢔ࣞࣝࢠ࣮ᛶࢆྵࡵࡓගᏳ඲ᛶࡢホ౯ࢶ࣮ࣝ࡜ࡋ࡚ ROS ࢔ࢵࢭ࢖ࢆ฼⏝ࡍࡿࡇ࡜ࡣጇᙜ࡜⪃࠼ࡽࢀࡿ(Onoue et al., perspnal communication)㸬
⾲ 7 ගẘᛶ㝜ᛶࡀሗ࿌ࡉࢀ࡚࠸ࡿග࢔ࣞࣝࢠ࣮ᛶ≀㉁ගẘᛶ㝜ᛶሗ࿌

In vivo assay (Guinea pigs)

In vitro assay㸦3T3 NRU-PT㸧

ග࢔ࣞࣝࢠ࣮ᛶ≀㉁࡜ࡋ࡚ሗ࿌
ࡉࢀ࡚࠾ࡾ㸪In
vivo and/or In vitroගẘᛶヨ㦂࡛㝜ᛶࢆ♧ࡍ໬ྜ≀㸦18 ≀㉁㸧

㝜ᛶ㸦10 ≀㉁㸧6-Methylcoumarin
Bithionol
Indomethacin
Ketprofen
Musk ambretteMusk xylene
Piroxicam
Sulfanilamide
Tribromosalicylanilide
Trichlorocarbanilide㝜ᛶ PIF < 2㸦5 ≀㉁㸧
Dichlorophene
Fenticlor
Hexachlorophene
Hydrochlorothiazide
Isoniazid

PIF: Photo irritation factor

28
34␲㝧ᛶ PIF > 2 and ൏ 5㸦3 ≀㉁㸧
Octyl dimethyl PABA
Omadine (Na salt)
Pyridoxine HCl

5 3Rs ࡬ࡢ㛵୚㸦ື≀⚟♴㠃࠿ࡽࡢጇᙜᛶ㸧
ROS ࢔ࢵࢭ࢖࡛ࡣ໬ྜ≀ࢆ཯ᛂᾮ࡟⁐ゎࡋ࡚ගࢆ↷ᑕࡋࡓ㝿࡟⏘⏕ࡉࢀࡿ ROS ࢆᐃࡍࡿヨ㦂⣔࡛࠶ࡾ㸪ື≀࠶ࡿ࠸ࡣື≀⏤᮶ࡢ⏕యᮦᩱࢆ౑⏝ࡋ࡞࠸ࡇ࡜࠿ࡽ㸪ROS ࢔ࢵࢭ࢖ࡢᐇ᪋࡟㝿ࡋ࡚ື≀⚟♴㠃ࡢၥ㢟ࡣᏑᅾࡋ࡞࠸㸬
ROS ࢔ࢵࢭ࢖ࡣ㸪2014 ᖺ 5 ᭶࡟බ㛤ࡉࢀࡓ་⸆ရࡢගᏳ඲ᛶ࢞࢖ࢻࣛ࢖ࣥ࡟࠾࠸࡚㸪ึᮇホ౯ࡢホ౯㡯┠ࡢࡦ࡜ࡘࡢ໬Ꮫⓗග཯ᛂᛶホ౯⣔࡜ࡋ࡚グ㍕ࡉࢀ㸪ROS ࢔ࢵࢭ࢖࡛㝜ᛶ࡜ุᐃࡉࢀࡓ㛤Ⓨ୰ࡢ་⸆ရ࡟㸪ࡉࡽ࡞ࡿගẘᛶヨ㦂ࢆ⾜࠺ᚲせ࡞࠸࡜ࡉࢀ࡚࠸ࡿ㸬ࡑࡢࡓࡵ㸪ROS ࢔ࢵࢭ࢖ࢆගᏳ඲ᛶホ౯࡟ᑟධࡍࡿࡇ࡜࡟ࡼࡾ㸪in vitro ගẘᛶヨ㦂࡛࠶ࡿ 3T3
NRU-PT ࡢヨ㦂ᩘࢆ๐ῶࡍࡿࡇ࡜ࡀ࡛ࡁ㸪ࡉࡽ࡟ in vivo ගẘᛶヨ㦂ࡢヨ㦂ᩘ๐ῶ࡟ࡘ࡞ࡀࡿ㸬

6 ヨ㦂᪉ἲࡢ᭷⏝ᛶ࡜ၥ㢟Ⅼ⣽⬊ࡸື≀ࢆ฼⏝ࡍࡿගᏳ඲ᛶホ౯⣔࡛ࡣ࢚ࢿࣝࢠ࣮ࡢ㧗࠸ UVB ࢆග※࡜ࡋ࡚౑⏝ࡍࡿࡇ࡜ࡀ㞴ࡋࡃ㸪ከࡃࡢሙྜࡣ UVA ࡢࡳࡢ↷ᑕࢆవ൤࡞ࡃࡉࢀࡿ㸬ࡇࡢࡓࡵ㸪UVB 㡿ᇦ࡟ࡋ࠿྾཰ࢆ᭷ࡉ࡞࠸໬ྜ≀࡟ᑐࡋ࡚ࡣഇ㝜ᛶホ౯ࢆ୚࠼ࡿࡇ࡜ࡀ࠶ࡿࡀ㸪ROS ࢔ࢵࢭ࢖ࡣࢩࣥࣉࣝ࡞≀ᛶホ౯⣔࡛࠶ࡿࡓࡵ࡟ UVB ࡢ↷ᑕࡶྍ⬟࡜࡞ࡿ㸬ࡲࡓ㸪⏕యヨᩱࢆᚲせ࡜ࡋ࡞࠸ࢩࢫࢸ࣒ࡢࡓࡵ㸪ࡑࡢ෌⌧ᛶࡶ㧗࠸㸬୍᪉㸪᪉ἲㄽⓗ࡞㝈⏺࡜ࡋ࡚ࡣ ROS ࢔ࢵࢭ࢖ࡣ໬ྜ≀ࢆ཯ᛂᾮ࡟⁐ゎࡋ࡚ගࢆ↷ᑕࡋࡓ㝿࡟⏘⏕ࡉࢀࡿ ROS 㔞ࢆ྾ගᗘࡢኚ໬࠿ࡽ
ᐃࡍࡿヨ㦂⣔࡛࠶ࡿࡓࡵ㸪཯ᛂᾮ୰࡛ᯒฟࡍࡿ⁐ゎᛶࡢప࠸≀㉁ࡸ⁐ゎ᫬࡟╔Ⰽࡀㄆࡵࡽ
ࢀࡿ໬Ꮫ≀㉁ࡣホ౯࡛ࡁ࡞࠸㸬ࡇࢀࡽㄢ㢟࡟ࡘ࠸࡚ࡢゎỴ⟇࡜ࡋ࡚ᨵⰋᆺ ROS ࢔ࢵࢭ࢖ࡸ
ヨ㦂ࢥࣥࢺ࣮ࣟࣝ⁐ᾮࡢタ⨨࡞࡝ࡀᥦ᱌ࡉࢀ࡚࠾ࡾ㸪ࡑࢀࡽ࡛ᚓࡽࢀࡓ▱ぢ࡟ࡘ࠸࡚ࡣ
ROS ࢔ࢵࢭ࢖ࡢ⤖ᯝุ᩿ࡢ᭦࡞ࡿຓࡅ࡟࡞ࡿ࡛࠶ࢁ࠺㸬ࡲࡓ㸪ROS ࢔ࢵࢭ࢖ࡣ௦ㅰ㐣⛬ࢆྵࡲ࡞࠸ࡇ࡜࠿ࡽ⿕㦂≀㉁ࡢ⏕యෆ௦ㅰ࡟౫ᏑࡍࡿගᏳ඲ᛶࣜࢫࢡࢆண

ࡍࡿࡇ࡜ࡀ㞴ࡋ࠸㸬ヨ㦂࡟⏝࠸ࡓ⃰ᗘ࡜㝧ᛶ/㝜ᛶุᐃ࡟ࡘ࠸࡚㸪㝧ᛶ⤖ᯝࡢุᐃ࡞ࡽࡧ࡟ 200 μM ⃰ᗘ࡛ࡢ㝜ᛶุᐃ࡟ࡣၥ㢟࡞࠸ࡀ㸪20 μM ⃰ᗘ࡛ࡢ㝜ᛶุᐃ࡟ࡣὀពࡍࡿᚲせࡀ࠶ࡿ㸬ࣂࣜࢹ࣮ࢩࣙࣥ
࣐ࢿࢪ࣓ࣥࢺࢳ࣮࣒ࡀ➨୕⪅ホ౯ጤဨ఍ࡢ່࿌ࢆཷࡅ࡚సᡂࡋࡓ᥎ዡࣉࣟࢺࢥ࣮࡛ࣝࡣᯒ
ฟ㸪╔Ⰽ࡟ࡼࡾ 200μM ุ࡛ᐃ࡛ࡁ࡞࠸ሙྜ࡟ 20 μM ࡢヨ㦂⤖ᯝࢆ᥇⏝࡛ࡁࡿ࡜ࡋ࡚࠸ࡿ㸬ࡋ࠿ࡋ࡞ࡀࡽ㸪⾜ᨻ࡬ࡢ⏦ㄳ᫬࡟ࡉࡽ࡞ࡿගẘᛶホ౯ࡣ୙せ࡜ุ᩿ࡍࡿ᰿ᣐ࡜ࡋ࡚ 20 μMࡢ㝜ᛶ⤖ᯝࢆ฼⏝ࡍࡿሙྜ࡟ࡣ㸪⿕㦂≀㉁ࡢග↷ᑕ᮲௳ୗ࡛ࡢᏳᐃᛶࢆ☜ㄆࡍࡿࡶࡢ࡜つᐃࡋ࡚࠸ࡿ㸬୍᪉㸪་⸆ရࡢගᏳ඲ᛶホ౯࢞࢖ࢻࣛ࢖ࣥ㸦ཌ⏕ປാ┬㸪2014㸧࡛ࡣ ROS ࢔ࢵࢭ࢖࡟㛵ࡋ࡚㸪ࠕ200μM ࡢヨ㦂⃰ᗘ࡛㐺ษ࡞᮲௳ୗ࡛ᐇ᪋ࡉࢀࡓሙྜ㸪ࡇࡢヨ㦂ἲ࡛ࡢ㝜ᛶ⤖ᯝࡣගẘᛶࡢᠱᛕࡀ㠀ᖖ࡟ప࠸ࡇ࡜ࢆ♧ࡍ͐ࠖ࡜グ㍕ࡉࢀ㸪20 μM ࡛ࡢ㝜ᛶุᐃ⤖ᯝࡣࡉࡽ࡞ࡿගᏳ඲ᛶヨ㦂ࢆ୙せ࡜ࡍࡿ᰿ᣐ࡜ࡋ࡚᥇⏝ࡋ࡞࠸ࡇ࡜ࢆ♧ࡋ࡚࠸ࡿ㸬ࣂࣜࢹ࣮

29
35

ࢩࣙࣥሗ࿌᭩࡟ࡼࡿ࡜㸪ࣉࣞࣂࣜࢹ࣮ࢩ࡛ࣙࣥࡢ㝧ᛶ໬Ꮫ≀㉁ 11 ≀㉁ࡣ㸪200 μM ࡛඲≀㉁ࡀ㝧ᛶࢆ♧ࡋ㸪20 μM ࡛ࡣ 10 ≀㉁ࡀ㝧ᛶ⤖ᯝࢆ♧ࡋ㸪1 ≀㉁㸦8-MOP㸧ࡀ㝜ᛶࢆ♧ࡋࡓ㸬ࡲࡓ㸪ICH ࡢ S10 E W G ࡢᣦ᦬ࢆཷࡅ࡚㸬ࣂࣜࢹ࣮ࢩࣙࣥཧຍ᪋タ 1 ᪋タ࡟࡚ᐇ᪋ࡋࡓࣂ
ࣜࢹ࣮ࢩࣙࣥ࡟⏝࠸ࡓ 42 ≀㉁࡟ࡘ࠸࡚㸪20 μM ⃰ᗘ࡛ࡢ ROS ࢔ࢵࢭ࢖ࢆᐇ᪋ࡋࡓ⤖ᯝࡣ200 μM ⃰ᗘᐃ᫬࡟ẚ࡭ࡿ࡜཯ᛂࡣᑠࡉ࠿ࡗࡓࡀ㸪ࡍ࡭࡚ࡢ㝧ᛶ≀㉁࡛㝧ᛶࢆ♧ࡋ㸪8-MOPࡶྵࡵ㸪ഇ㝜ᛶ⤖ᯝࢆ࿊ࡋࡓ≀㉁ࡣ࡞࠿ࡗࡓ㸬200 μM ࡟ẚ࡭㸪20 μM ࡛཯ᛂࡀᑠࡉࡃ࡞ࡿ
⃰ᗘ࡟㛵㐃ࡋࡓせᅉ࡜ࡋ࡚㸪໬ྜ≀ࡢගᏳᐃᛶࡀ⪃࠼ࡽࢀࡓࡇ࡜㸪࡞ࡽࡧ࡟㸪20 μM ࡛ࡢഇ㝜ᛶ⤖ᯝࢆ♧ࡋࡓ 8-MOP ࡣ 200 μM ⃰ᗘ࡛ホ౯ྍ⬟࡛㝧ᛶ⤖ᯝࢆ♧ࡋࡓࡇ࡜࠿ࡽ㸪᥎ዡࣉࣟࢺࢥ࣮࡛ࣝࡣ 20μM ⃰ᗘࡢ⤖ᯝࡣ 200 μM ࡛ホ౯࡛ࡁ࡞࠸ሙྜ࡛㸪࠿ࡘ㸪 ᐃ᮲௳ୗ࡛⿕㦂≀㉁࡟ග࡟ࡼࡿศゎࡀㄆࡵࡽࢀ࡞࠸ሙྜ࡟㝈ᐃࡍࡿࡇ࡜࡟ࡼࡾ㸪ࣂࣜࢹ࣮ࢩࣙࣥࡢ⤖ᯝࡀṇ☜࡟཯ᫎࡉࢀ࡚࠸ࡿ㸬୍᪉㸪ICH S10 EWG ࡛ࡣ 20 μM ࡛ࡢ㝜ᛶ⤖ᯝࢆ་⸆ရ㛤ⓎရࡢගᏳ඲ᛶホ౯ࢆ୙せ࡜ࡍࡿุ᩿ࡢ᰿ᣐ࡜ࡋ࡚᥇⏝ࡍࡿࡇ࡜࡟ៅ㔜࡛࠶ࡾ㸪㝜ᛶ⤖ᯝࡣ 200
μM ࡛ࡢヨ㦂⤖ᯝࡢࡳࢆ᥇⏝ࡍࡿࡇ࡜࡜࡞ࡗࡓ㸬௒ᚋ㸪20 μM ⃰ᗘ࡛ࡢヨ㦂⤖ᯝࡢ㞟✚࠶ࡿ
࠸ࡣ 200 μM ⃰ᗘ࡛ࡢྍ⁐໬➼ࡢᯒฟ㜵Ṇᢏ⾡ࡢᛂ⏝࡟ࡼࡾ㸪ヨ㦂⤖ᯝࡢ฼⏝᪉ἲࡀ」ᩘ࠶
ࡿࡇ࡜࡟ࡼࡿΰ஘ࡀゎᾘࡉࢀࡿࡇ࡜ࡀᮃࡲࢀࡿ㸬ΰྜ≀࠾ࡼࡧศᏊ㔞୙᫂ヨᩱ࡟ࡘ࠸࡚ࡣ㸪μg/mL ༢఩࡛ࡢ ROS ࢔ࢵࢭ࢖࡟㛵ࡍࡿሗ࿌ࡀ
࡞ࡉࢀ࡚࠸ࡿ㸬་⸆ရ࡛ࡣ ROS ࢔ࢵࢭ࢖ࡣගᏳ඲ᛶࡢึᮇホ౯࡜ࡋ࡚㸪ගᏳ඲ᛶホ౯ࡢせྰุ᩿࡟฼⏝ྍ⬟࡛࠶ࡾ㸪ICH ຍ┕ᅜࡢつไᙜᒁ࡟ࡼࡾཷࡅධࢀࡽࢀࡿ㸬໬Ꮫ≀㉁࠾ࡼࡧ㎰⸆࡛ࡣ㸪ROS ࢔ࢵࢭ࢖ࡣග཯ᛂᛶࢆホ౯ࡍࡿࡇ࡜࡛㸪₯ᅾⓗ࡞ගẘᛶࡢࣁࢨ࣮ࢻ᳨ฟࡢᡭἲ࡜ࡋ࡚᭷⏝࡛࠶ࢁ࠺㸬໬⢝ရ࡛ࡣ㸪ROS ࢔ࢵࢭ࢖࠾ࡼࡧᨵⰋᆺ ROS ࢔ࢵࢭ࢖ࢆ㐺⏝ࡍࡿࡇ࡜࡛㸪ගẘᛶ㸪ග࢔ࣞࣝࢠ࣮ᛶ≀㉁ࡢ᳨ฟࡀྍ⬟࡛࠶ࡿࡇ࡜ࡀ♧ࡉࢀ࡚࠸ࡿ㸦Onoue et al., 2013㸧㸬ࡋ࠿ࡋ࡞ࡀ
ࡽ㸪ග໬Ꮫⓗ཯ᛂᛶヨ㦂࡛࠶ࡿ ROS ࢔ࢵࢭ࢖ࡢ≉ᛶ࠿ࡽ㸪㐺ษ࡟ᐇ᪋ࡉࢀࡓ ROS ࢔ࢵࢭ࢖࡛㝜ᛶ࡛࠶ࢀࡤගẘᛶ㸪ග࢔ࣞࣝࢠ࣮ᛶࢆ♧ࡉ࡞࠸࡜ุ᩿ࡋ࡚ᕪᨭ࠼࡞࠸ࡀ㸪ROS ࢔ࢵࢭ࢖࡟࠾ࡅࡿ㝧ᛶࡣග཯ᛂᛶࢆ᭷ࡍࡿࡇ࡜ࢆ♧ࡍࡶࡢ࡛࠶ࡗ࡚㸬ගẘᛶ㸪ග࢔ࣞࣝࢠ࣮ᛶࡢ
᭷↓ࡢホ౯ࡣู㏵ホ౯ࡉࢀࡿ࡭ࡁ࡛࠶ࡿ㸬

7 ⤖ㄽ
ROS ࢔ࢵࢭ࢖ࡣ໬Ꮫ≀㉁࡟ᨃఝኴ㝧ගࢆ↷ᑕࡋ㸪singlet oxygen ࡜ super oxide anion ࡢ⏘⏕ࡢ᭷↓ࢆㄪ࡭ࡿග཯ᛂᛶヨ㦂ࡢ୍ࡘ࡛࠶ࡿ㸬7 ᪋タ࡟ࡼࡾᐇ᪋ࡉࢀࡓከ᪋タࣂࣜࢹ࣮ࢩࣙ
ࣥヨ㦂࡟࠾࠸࡚㸪ROS ࢔ࢵࢭ࢖ࡣ㸪᪋タෆࡢ᪥ෆᕪ/᪥㛫ᕪ࠾ࡼࡧ᪋タ㛫ᕪࡀᑠࡉࡃ㸪ỗ⏝ᛶ࡟ඃࢀ࡚࠸ࡿヨ㦂ἲ࡛࠶ࡿࡇ࡜ࡀ♧ࡉࢀࡓ㸬ࡲࡓ㸪㞴⁐ᛶࡢࡓࡵホ౯࡛ࡁ࡞࠿ࡗࡓ⿕㦂≀㉁ࢆ㝖ࡃ࡜㸪ROS ࢔ࢵࢭ࢖࡟ࡼࡿගẘᛶ≀㉁ࡢ㝧ᛶ᳨ฟ⋡ࡣ 100%࡛࠶ࡾ㸪ഇ㝜ᛶ⤖ᯝࡀ࡞࠸ࡇ࡜࠿ࡽ㸪ROS ࢔ࢵࢭ࢖ࡣගẘᛶ࣏ࢸࣥࢩࣕࣝࡢホ౯࡟᭷⏝࡛࠶ࡿ࡜⪃࠼ࡽࢀࡓ㸬30
36ࡉࡽ࡟ ROS ࢔ࢵࢭ࢖ከ᪋タࣂࣜࢹ࣮ࢩࣙࣥࡢ➨୕⪅ホ౯ጤဨ఍ࡣࡑࡢሗ࿌᭩㸦Spielmann
et al., 2013㸧࡟࠾࠸࡚㸪ࠕጤဨ఍ࡣ㸪ROS ࢔ࢵࢭ࢖ࡢ෌⌧ᛶ࠾ࡼࡧண

ᛶࡀ୍㐃ࡢගᏳ඲ᛶ

ヨ㦂ᐇ᪋࡞ࡽࡧ࡟་⸆ရࡢ◊✲㛤Ⓨ࡟࠾ࡅࡿពᛮỴᐃᡓ␎࡛ ROS ࢔ࢵࢭ࢖ࡀ฼⏝ࡉࢀࡿࡇ࡜ࢆᨭᣢࡍࡿࡇ࡜࡟༑ศ࡛࠶ࡿ࡜⤖ㄽࡍࡿ㸬ࡇࡢᡓ␎࡟࠾࠸࡚㸪ROS ࢔ࢵࢭ࢖࡛㝧ᛶ㸪ᙅ࠸㝧ᛶࢆ♧ࡍ࠿㸪ุᐃ୙⬟ࡢሙྜ࡟ࡣ㸪3T3 NRU-PT (OECD Test Guideline 432)࡞࡝ࡢḟࡢࣞ࣋ࣝࡢ in vitro ヨ㦂⣔࡟㐍ࡵ㸪㝜ᛶࡢሙྜ࡟ࡣື≀࡛ࡢヨ㦂ࡸ௚ࡢヨ㦂ࡣせồࡉࢀ࡞࠸㸬ጤဨ఍ࡣࡲࡓ㸪ROS ࢔ࢵࢭ࢖ࡣගᏳ඲ᛶホ౯࡟せࡍࡿᮇ㛫㸪㈝⏝࠾ࡼࡧ౑⏝ື≀ᩘࡢ๐ῶ࡟ࡘ࡞ࡀࡿ࡜⤖ㄽࡋࡓ㸬ࡉࡽ࡟ ROS ࢔ࢵࢭ࢖ࢆගᏳ඲ᛶホ౯ᡓ␎࡟⤌ࡳධࢀࡿࡇ࡜ࡣ 3T3
NRU-PT ࡸࡑࢀ࡟⥆ࡃື≀࡛ࡢヨ㦂ࡀᚲせ࡜࡞ࡿ໬ྜ≀ᩘࢆ᭷ព࡟ῶᑡࡉࡏࡿࡇ࡜࡟ࡘ࡞
ࡀࡿ㸬ࠖ࡜⤖ㄽࡋࡓ㸬ᮏࣂࣜࢹ࣮ࢩࣙࣥ⤖ᯝ࡟ᇶ࡙ࡁ㸪ICH ࡢගᏳ඲ᛶホ౯࢞࢖ࢻࣛ࢖ࣥ࡟࠾࠸࡚㸪ROS ࢔ࢵ
ࢭ࢖ࡣ඲㌟࠾ࡼࡧ⓶⭵㐺⏝⸆࡟࠾ࡅࡿගẘᛶホ౯ࡢせྰุ᩿࡟฼⏝ྍ⬟࡞ヨ㦂ἲࡢ୍ࡘ࡜
ࡋ࡚᥇ᢥࡉࢀࡓ㸦Step 4㸪2013 ᖺ 11 ᭶㸧㸬௒ᚋ㸪ROS ࢔ࢵࢭ࢖࡛㝜ᛶࡢሙྜࡣ㸪ࡉࡽ࡞ࡿගẘᛶホ౯ࡀ୙せ࡜࡞ࡾ㸪ගẘᛶヨ㦂ᩘ࡜ࡑࡢ㈝⏝ࡢ๐ῶຠᯝࡀᮇᚅࡉࢀࡿ㸬ࡉࡽ࡟᭱㏆ࡢ᳨ウ࡟࠾࠸࡚㸪ග࢔ࣞࣝࢠ࣮ᛶ≀㉁ࡶ ROS ࢔ࢵࢭ࢖᳨࡛ฟࡍࡿࡇ࡜ࡀ࡛ࡁࡿ࡜ࡢ▱ぢࡀᚓࡽࢀ㸪ROS ࢔ࢵࢭ࢖ࡣගẘᛶࡔࡅ࡛࡞ࡃ㸪ග࢔ࣞࣝࢠ࣮ᛶࡶྵࢇࡔගᏳ඲ᛶࡢホ౯ࢶ࣮ࣝ࡜ࡋ࡚฼⏝ࡉࢀࡿࡇ࡜ࡀᮇᚅࡉࢀࡿ㸬໬⢝ရཎᩱ࡟ࡘ࠸࡚ΰྜ≀࠾ࡼࡧศᏊ㔞୙᫂ヨᩱ࡬ࡢ㐺⏝ྍ⬟ᛶࡀ♧ࡉࢀ㸪໬⢝ရཎᩱ㸪୍⯡໬Ꮫ≀㉁࡞ࡽࡧ࡟㎰⸆࡟ࡘ࠸࡚
ROS ࢔ࢵࢭ࢖ࡀ₯ᅾⓗ࡞ගᏳ඲ᛶ࡟㛵ࡍࡿࣁࢨ࣮ࢻ᳨ฟࡢᡭἲ࡜ࡋ࡚᭷⏝࡛࠶ࡿࡇ࡜ࡀᮇᚅࡉࢀࡿ㸬

8 ⏝ㄒࡢゎㄝ
Chromophore㸦ࢡࣟࣔࣇ࢛࢔㸧㸸ྍどග࠶ࡿ࠸ࡣ UV ࢆ྾཰ࡍࡿศᏊࡢ㒊ศᵓ㐀㸬
Internal conversion㸦ෆ㒊㌿⛣㸧㸸࠶ࡿศᏊࡀࡼࡾ࢚ࢿࣝࢠ࣮ࡢ㧗࠸㸪ࡶࡋࡃࡣప࠸᣺ືບ㉳≧ែ࡟㑄⛣ࡍࡿࡇ࡜㸬Intersystem crossing㸦㡯㛫஺ᕪ㸧㸸㧗࠸᣺ືບ㉳≧ែࡢศᏊ࠿ࡽࡢࢫࣆࣥኚ᥮࡟కࡗ࡚㏆ࡃ࡟ᏑᅾࡍࡿศᏊࡀ␗࡞ࡿ᣺ືບ㉳≧ែ࡟㑄⛣ࡍࡿࡇ࡜㸬

9 ཧ⪃ᩥ⊩
Andersen KE, Lindskov R (1984) Recall of UVB-induced erythema in breast cancerpatient receiving multiple drug
chemotherapy. Photodermatol, 1, 129-132.
Bauer D, Averett LA, De Smedt A, Kleinman MH, Muster W, Pettersen BA, Robles C (2014), Standardized UV-vis
spectra as the foundation for a threshold-based, integrated photosafety evaluation. Regul Toxicol Pharmacol, 68(1),70-75.

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Boccumini LE, Fowler CL, Campbell TA, Puertolas LF, Kaidbey KH (2000) Photoreaction potential of orally
administered levofloxacin in healthy subjects. Ann Pharmacother, 34(4), 453-458.
Dam C, Bygum A (2008) Subacute cutaneous lupus erythematosus induced or exacerbated by proton pump inhibitors.
Acta Derm Venereol, 88(1), 87-89.
Dillaha CJ, Jansen GT, Honeycutt WM, Bradford AC (1983) Selective cytotoxic effect of topical 5-fluorouracil. Arch
Dermatol, 119(9), 774-783.
Henry B, Fotib C, Alsanteb K (2009) Can light absorption and photostability data be used to assess the photosafety
risks in patients for a new drug molecule? J Photochem Photobiol B, 96(1), 57-62.
Kirkup ME, Narayan S, Kennedy CTC (2003) Cutaneous recall reactions with systemic fluorouracil. Dermatology,
206(2), 175-176.
Kleinman MH, Smith MD, Kurali E, Kleinpeter S, Jiang K, Zhang Y, Kennedy-Gabb SA, Lynch AM, Geddes CD(2010) An evaluation of chemical photoreactivity and the relationship to phototoxicity. Regul Toxicol Pharmacol,
58(2), 224-232.
Ljunggren B, Sjövall P (1986) Systemic quinine photosensitivity. Arch Dermatol, 122(8), 909-911.
Miolo G, Marzano C, Gandin V, Palozzo AC, Dalzoppo D, Salvador A, Caffieri S (2011) Photoreactivity of
5-Fluorouracil under UVB Light. Chem Res Toxicol, 24(8), 1319–1326.
Nishida H, Hirota H, Seto Y, Suzuki G, Kato M, Kitagaki M, Sugiyama M, Kouzuki H, Onoue S (2015) Non-animal
photosafety screening for complex cosmetic ingredients with photochemical and photobiochemical assessment tools.
Regul Toxicol Pharmacol, 72(3):578-585.
Onoue S, Tsuda Y (2006) Analytical studies on the prediction of photosensitive/phototoxic potential ofpharmaceutical substances. Pharm Res 23(1), 156-164Onoue S, Igarashi N, Yamada S, Tsuda Y (2008a)
High-throughput reactive oxygen species (ROS) assay: an enabling technology for screening the phototoxic potentialof pharmaceutical substances. J Pharm Biomed Anal, 46(1), 187-193.
Onoue S, Yamauchi Y, Kojima T, Igarashi N, Tsuda Y (2008b) Analytical studies on photochemical behavior ofphototoxic substances; Effect of detergent additives on singlet oxygen generation. Pharm Res, 25(4), 861–868.
Onoue S, Seto Y, Gandy G, Yamada, S (2009) Drug-Induced Phototoxicity; An Early In Vitro Identification of
Phototoxic Potential of New Drug Entities in Drug Discovery and Development. Curr Drug Saf, 4(2), 123-136.
Onoue S, Ochi M, Gandy G, Seto Y, Igarashi N, Yamauchi Y, Yamada S (2010) High-throughput screening system
for identifying phototoxic potential of drug candidates based on derivatives of reactive oxygen metabolites. Pharm
Res, 27(8), 1610-1619.
Onoue S, Hosoi K, Wakuri S, Iwase Y, Yamamoto T, Matsuoka N, Nakamura K Toda T, Takagi H, Osaki N,
MatsumotoY, Kawakami S, Seto Y, Kato M, Yamada S, Ohno Y and Kojima H (2013a) Establishment andintra-/inter-laboratory validation of a standard protocol of reactive oxygen species assay for chemical photosafety
evaluation. J Appl Toxicol, 33(11), 1241–1250.
Onoue S, Suzuki G, Kato M, Hirota M, Nishida H, Kitagaki M, Kouzuki H, Yamada S (2013b) Non-animal
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Nakamuraj K, Ohno Y, Kojima H (2014a) Intra-/inter-laboratory validation study on reactive oxygen species assay
for chemical photosafety evaluation using two different solar simulators, Toxicol In Vitro, 28(4), 515–523
Onoue S, Kato M, Yamada S (2014b) Development of an albuminous reactive oxygen species assay for photosafety
evaluation under experimental biomimetic conditions, J Appl Toxicol, 34(2), 158–165
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http://www.jacvam.jp/files/news/ROS_Assay_full%2020130920%20atlas_fourth%20data.pdf (accessed May,
2015)ROS assay validation management team (2013b) Validation report for the international validation study on
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evaluation of poorly water-soluble chemicals. Toxicol In Vitro, 27(6), 1838–1846
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WW, Maurer T, Moldenhauer F, Moore L, Pape WJ, Pfanenbecker U, Potthast J, De Silva O, Steiling W, Willshaw A.
(1994) EEC/COLIPA project on in vitro phototoxicity testing: First results obtained with a Balb/c 3T3 cell
phototoxicity assay. Toxicol In Vitro., 8(4), 793-796.
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Gerberick F, Liebsch M, Lovell WW, Maurer T, Pfannenbecker U, Potthast JM, Csato M, Sladowski D, Steiling W,
Brantom P. (1998a) The International EU/COLIPA In Vitro Phototoxicity Validation Study: Results of Phase II(Blind Trial). Part 1: The 3T3 NRU Phototoxicity Test. Toxicol In Vitro, 12(3), 305-327.
Spielmann H, Stokes WS, Horii I, Kim BH (2013) Peer Review Panel Evaluation of the Reactive Oxygen Species(ROS) Photosafety Assay, http://www.jacvam.jp/files/doc/02_03/02_03_C1.pdf (accessed May, 2015)
Tokura Y (2009) Photoallergy. Expert Rev Dermatol, 4(3), 263-270.

ᑿୖㄔⰋ㸪୰ᮧ࿴ᕷ㸦2011㸧 ගẘᛶ㸪♫ᅋἲே᪥ᮏ⸆⌮Ꮫ఍⦅㞟ࠕᐇ㦂⸆⌮Ꮫ ๰⸆◊✲ࡢࢫࢺࣛࢸࢪ࣮
ୗࠖ㸦㔠ⰾᇽ㸧ཌ⏕ປാ┬㸦2010㸧 㔜⠜๪స⏝⑌ᝈูᑐᛂ࣐ࢽࣗ࢔ࣝ ᡭ㊊⑕ೃ⩌㸦http://www.info.pmda.go.jp/juutoku/file/jfm1003014.pdf㸪2015 ᖺ 2 ᭶࢔ࢡࢭࢫ☜ㄆ㸧ཌ⏕ປാ┬㸦2014㸧 ་⸆ရࡢගᏳ඲ᛶホ౯࢞࢖ࢻࣛ࢖ࣥ࡟ࡘ࠸࡚㸦⸆㣗ᑂᰝⓎ 0521 ➨ 1 ྕ㸪ᖹᡂ 26 ᖺ 5
᭶ 21 ᪥㸧

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10 Appendix
Appendix 1 Executive Summary of “Peer Review Panel
Evaluation of the Reactive Oxygen Species (ROS) Photosafety
Assay” (16 October 2013)
Executive Summary
The Japanese Center for the Validation of Alternative Methods (JaCVAM) convened an
independent scientific peer review panel to evaluate the validation status of the Reactive
Oxygen Species (ROS) assay in accordance with established international criteria (OECD,2005). The ROS assay is a test method proposed as a component of an integrated
photosafety testing strategy to evaluate whether test substances such as pharmaceuticals
have the potential to cause phototoxicity.
The panel met initially in February and again in August 2013 in Tokyo, Japan. The panel
considered the reports of two international validation studies and a proposed ROS ssay
protocol at their initial meeting. The panel subsequently reviewed updated versions of the
ROS Assay protocol and the validation study reports as revised by the Validation
Management Team (VMT). This report summarizes the panel’s final evaluation andconclusions.
Overall conclusion: The panel concluded that the reproducibility and predictivity of the
ROS assay is sufficient to support its use in an integrated photosafety testing and decision
strategy for drug research and development. In this strategy, negative results in the ROS
assay would not require further testing in animals or other tests, while positive, weakly
positive, and inconclusive results would proceed to the next level of testing in an in vitro test
system such as the 3T3 Phototoxicity Assay (OECD Test Guideline 432). The panel also
concluded that use of the ROS assay could potentially provide significant savings in time,
cost and reduced animal use for photosafety assessments. Furthermore, incorporating the
ROS assay into a photosafety testing strategy is expected to significantly reduce the overall
number of substances that would
require additional testing in the in vitro 3T3 Phototoxicity Assay and subsequent testing inanimals.
Regulatory rationale: The panel concluded that the ROS Assay is applicable for use within
the ICH regulatory testing framework for photosafety evaluation of pharmaceutical products.
Regulatory authorities (e.g. PMDA/MHLW, U.S. FDA, EMEA) require non-clinicalphotosafety testing prior to approving First-in-Human Phase I studies so that appropriate
precautions and observations can be taken during initial human studies. Such non-clinicalphotosafety testing typically includes an assessment of the potential for a drug to cause
phototoxic reactions, which are characterized clinically by dermal redness, swelling, irritation,and inflammation. The panel
also agreed that the ROS assay is applicable to in-house drug research and development.
Scientific rationale: The panel recognized that ROS production is the most importantmechanism for chemically-induced phototoxicity, and is therefore a critical pathway initiating
event leading to phototoxicity. The ROS assay quantitatively measures two common
reactive oxygen species generated by photoreactive chemicals after exposure to simulated
sunlight. In this validation study, chemicals that did not produce sufficient ROS to meet thephotoreactivity threshold classification criteria for the ROS assay are uniformly
non-phototoxic, while chemicals that met or exceeded the photoreactivity classification
criteria include all known phototoxicants. Therefore if a chemical is not photoreactive in the

34
40

ROS assay, it is unlikely that phototoxicity will occur in living systems.
Limitations: The panel noted that the ROS assay assesses chemical photoreactivity in a
nonbiological system, and therefore may overpredict phototoxicity potential since it does notassess the direct interaction of chemicals with biological tissues. The assay may also
overestimate the skin for phototoxic reactions to occur, or photodegradation may occur.
Accordingly, positive results in the ROS assay are generally recommended for further
evaluation in a photosafety testing strategy.
Validation study reference chemicals: The panel agreed that the reference chemicals
selected for the validation studies were appropriate and sufficiently representative of thechemicals likely to be evaluated in the assay. The 42 reference chemicals incorporated most
known human phototoxicants and included 23 known positives and 19 negatives. The
chemicals were backed by data from human patch testing and in vitro 3T3 phototoxicity
assay results. All data from the validation studies were made available in the validation
study reports.
Assay Reproducibility: The panel concluded that the assay had excellent reproducibility
both within and between laboratories for the 42 reference chemicals evaluated in thevalidation studies. Additionally, the positive and negative control chemicals had 100%
reproducibility within and between laboratories based on classification outcome, whichfurther supports the reproducibility of the ROS assay.
Test method predictivity: After reviewing analyses provided in the validation study reports,
the panel agreed that conducting a single assay per chemical provided optimal predictivity.
The panel concluded that the classification criteria for test outcomes have been
appropriately optimized to avoid false negatives while minimizing false positives. The panel
also noted that chemicals positive for both reactive oxygen species were uniformly
phototoxic.
Data quality: The panel agreed that the high level of within and between laboratory
reproducibility suggested a consistently high level of quality of the validation studies. While
the studies were not conducted in strict accordance with GLPs, most of the labs were GLP
certified. The validation management team also confirmed that quality control audits foundthat validation report data accurately reflected the raw data results.
Test method protocols: The panel considered the test method protocols used for the twovalidation studies and key aspects of a proposed standardized ROS assay protocol. The
panel recommended that the solar simulator should be equipped with an appropriate
temperature control unit or fan since ROS production can be influenced by temperature. The
panel concluded that the list of proficiency chemicals provided in the test method protocol
for laboratories to use to demonstrate ability to perform the assay was appropriate. The
panel recommended that each lab should develop historical positive and negative control
value acceptance ranges that can be used to determine the acceptability of an individual
test. The panel also agreed with the appropriateness of the reference chemicals identified
for qualification of solar simulators other than the two used in the validation studies.
Applicability domain: The applicability domain of the ROS assay is currently restricted toonly those chemicals that meet the solubility criteria outlined in the protocol. The panel
recommended that as experience is gained from use of the ROS assay, the applicability
domain could be more fully described in terms of physicochemical properties and/or
chemical classes. This will contribute to increased efficiency by providing criteria that can beused to identify whether a chemical may be satisfactorily tested in the ROS assay, or
whether an alternate assay should be used initially.

35
41

Appendix 2 List of reasons for chemical selection
NO.

CompoundReasons for chemical selection
5-FU was reportedly a phototoxic chemical in humans [Dillaha et al., 1983], but it
was negative in the 3T3NRU-PT [Kleinman et al., 2010 and Onoue et al., 2010].
Reported causes of human phototoxicity in 5-FU include photocytotoxicity induced
by UV-B alone [Kirkup M.E. et al., 2003 and Andersen K.E. et al., 1984] and/or
ROS generation derived from UV-B induced photodegradation [Miolo G. et al.,2011]. 5-FU absorbs mainly UV-B (290–320 nm) within the range of natural
sunlight (Appendix 7), UV-B irradiation might be essential for photochemical
activation of 5-FU.

Therefore it was unknown whether 5-FU was a phototoxic

chemical, and high quality human data was not available. 5-FU was selected inorder to provide information on the limits of the ROS assay. Photosensitivity was
I-1

5-Fluorouracil (5-FU)

mentioned on both the US and the JPN label.
8-MOP was selected from the list of phototoxic chemicals used in the 3T3NRU-PT
validation study. Human data for this chemical was described as positive in thearticle on the 3T3 NRU-PT validation study [Spielmann et al, 1994a].

I-2

8-Methoxy psoralen (8-MOP)

Photosensitivity was mentioned on both the US and the JPN label.
Amiodarone HCl was selected from the list of phototoxic chemicals used in the
3T3NRU-PT validation study. Human data for this chemical was described as
positive in the article on the 3T3 NRU-PT validation study [Spielmann et al,

I-3

Amiodarone HCl

1994a].

Photosensitivity was mentioned on both the US and the JPN label.

Chlorpromazine HCl was selected from the list of phototoxic chemicals used in the
3T3NRU-PT validation study. Human data for this chemical was described as
positive in the article on the 3T3 NRU-PT validation study [Spielmann et al,I-4

Chlorpromazine HCl

1994a].

Photosensitivity was mentioned on both the US and the JPN label.

Diclofenac was selected as a phototoxic chemical for human per the article byPrzybilla et al [1987].
I-5

Diclofenac

Photosensitivity was mentioned on both the US and the

JPN label.
Doxycycline HCl was selected from the list of phototoxic chemicals used in the
3T3NRU-PT validation study. Human data for this chemical was described as
positive in the article on the 3T3 NRU-PT validation study [Spielmann et al,

I-6

Doxycycline HCl

1994a].

Photosensitivity was mentioned on both the US and the JPN label.

Furosemide was selected from the list of phototoxic chemicals used in the
I-7

Furosemide

3T3NRU-PT validation study. Human data for this chemical was described as3642

positive in the article on the 3T3 NRU-PT validation study [Spielmann et al,
1998a].

Although the 2011 US label did mention photosensitivity, 2012 US label

did not. Photosensitivity was mentioned on the JPN label.
Ketoprofen was selected from the list of phototoxic chemicals used in the
3T3NRU-PT validation study. Human data for this chemical was described as
positive in the article on the 3T3 NRU-PT validation study [Spielmann et al,I-8

Ketoprofen

1998a].

Photosensitivity was mentioned on both the US and the JPN label.

Levofloxacin was selected as a phototoxicity positive chemical for human per thearticle by Boccumini et al [2000].
I-9

Levofloxacin

Photosensitivity was mentioned on both the US

and the JPN label.
Norfloxacin was selected from the list of phototoxic chemicals used in the
3T3NRU-PT validation study. Human data for this chemical was described as
positive in the article on the 3T3 NRU-PT validation study [Spielmann et al,

I-10

Norfloxacin

1998a].

Photosensitivity was mentioned on both the US and the JPN label.

Omeprazole was selected as a phototoxic chemical for human per to the article by
Dam et al [2008].
I-11

Omeprazole

Photosensitivity was mentioned on both the US and the JPN

label.
Quinine HCl was used as positive control according to the method of Onoue et al.
[2008a].

Quinine HCl was classified as a phototoxic chemical for human per thearticle by Ljunggren et al [1986].
I-12

Quinine HCl

There was a mention of the photosensitivity in

the US label.
Sulizobenzone was used as negative control according to the method of Onoue etal. [2008a].

Sulisobenzone was classified as a non-phototoxic chemical, because

Human data for this chemical was described as negative in the article on the 3T3
I-13

Sulisobenzone

NRU-PT validation study [Spielmann et al, 1998b].

37
43

Appendix 3 Test chemicals for the Phase 1 study
UV/vis absorption b)

No.

Chemical name

CAS No.a)

MEC

Ȝmax

(L/mol/cm)(nm)

in vivo

ROS

3T3

assay

NRU

Animal

Human

I-1

5-FU

51-21-81800 c)

290 d)- 1)- 2, 3)

NA

+? 4)

I-2

8-MOP

298-81-7

3631

300

+ 1)

+ 5)

+ 5)

+ 5)

I-3

Amiodarone HCl

19774-82-4

5400

290 d)

+ 3)

+ 5)

+ 5)

+ 5)

I-4

Chlorpromazine HCl

69-09-0

1746

304

+ 1)

+ 5)

+ 5)

+ 5)

I-5

Diclofenac

15307-79-6

7800 c)

290 d)

+ 1)

+ 3)

+ 6)

+ 7)

I-6

Doxycycline HCl

10592-13-93715

290 d)

+ 1)

+ 5)

+ 5)

+ 5)

I-7

Furosemide

54-31-92650

290 d)+ 1)+/- 3,8,9)

NA

+ 8)

I-8

Ketoprofen

22071-15-4

2092

290 d)

+ 1)

+ 8)

- 8)

+ 8)

I-9

Levofloxacin

100986-85-4

13000 c)

333

+ 10)

+ 10)

+ 11)

+ 12)

I-10

Norfloxacin70458-96-7

3562

323

+ 1)

+ 3)

+ 8)

+ 8)

I-11

Omeprazole

73590-58-6

15000 c)

301

+ 1)

+/- 3)

NA

+ 13)

I-12

Quinine HCl

6119-47-7

1938

330

+ 1)

+ 3)

+ 6)

+ 14)

I-13

Sulisobenzone

4065-45-6

3519

290 d)

- 1)

- 3)

NA

- 15)

5-FU: 5-fluorouracil, 8-MOP䠖8-methoxy psoralen+ : Positive, - : Negative, +/- : Equivocal, NA : Not available, ? : uncleara) CAS No.: Chemical abstracts service registry number, b) The UV/vis absorbance (290-700 nm) of chemicals wasmeasured in 20 mM phosphate buffer (pH 7.4). Test chemicals were dissolved in dimethylsulfoxide (DMSO) at 10mM and diluted with 20 mM phosphate buffer (pH 7.4). Final concentration of DMSO was unified to 0.5%. c) Molar
extinction coefficient (MEC) of 5-FU, diclofenac, levofloxacin, and omeprazole were extracted from the articles ofOnoue et al. (2008a) and Seto et al. (2011).
d) Ȝmax (nm) was a wavelength at which the UV/vis absorbance shows a peak between 290 and 700 nm. Ȝmax (nm)
was indicated as 290 nm in the case where the peak absorption is located below 290 nm and the maximum absorption
is at 290 nm.
1) Onoue et al., 2008a, 2) Kleinman et al., 2010, 3) Onoue et al., 2010, 4) Dillaha et al., 1983, 5) Spielmann et al.,1994a, 6) Spielmann et al., 1994b, 7) Przybilla et al., 1987, 8) Spielmann et al., 1998a, 9) Peters et al., 2002, 10) Setoet al., 2011, 11) Wagai et al., 1992, 12) Boccumini et al., 2000, 13) Dam et al., 2008, 14) Ljunggren et al., 1986, 15)
Spielmann et al., 1998b38
44

Appendix 4 List of reasons for chemical selection
NO.

Chemical nameReasons for chemical selection

Phototoxic drugs
Acridine was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on
the 3T3 NRU-PT validation study [Spielmann et al, 1998a].
Acridine (No. II-1) and acridine HCl (No. II-2) were tested both as a free form and a salt
in order to test whether the aqueous solubility of chemicals might limit the predictive
II-1

Acridine

power of the ROS assay.
Acridine HCl was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on
the 3T3 NRU-PT validation study [Spielmann et al, 1998a].
Acridine (No. II-1) and acridine HCl (No. II-2) were tested both as a free form and a salt
in order to test whether the aqueous solubility of chemicals might limit the predictive

II-2

Acridine HCl

power of the ROS assay.
Amiodarone HCl was selected from the list of positive chemicals used in the 3T3
NRU-PT validation study. Human data for this chemical was described as positive in thearticle on the 3T3 NRU-PT validation study [Spielmann et al, 1994a].

II-3

Amiodarone HCl

Photosensitivity

was mentioned on both the US and the JPN label.
Chlorpromazine HCl was selected from the list of positive chemicals used in the 3T3
NRU-PT validation study. Human data for this chemical was described as positive in thearticle on the 3T3 NRU-PT validation study [Spielmann et al, 1994a].

II-4

Chlorpromazine HCl

Photosensitivity

was mentioned on both the US and the JPN label.
Doxycycline HCl was selected from the list of positive chemicals used in the 3T3
NRU-PT validation study. Human data for this chemical was described as positive in thearticle on the 3T3 NRU-PT validation study [Spielmann et al, 1994a].

II-5

Doxycycline HCl

Photosensitivity

was mentioned on both the US and the JPN label.
Fenofibrate was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on
the 3T3 NRU-PT validation study [Spielmann et al, 1998a].

Although the 2011 USlabel did mention photosensitivity, 2012 US label did not. Photosensitivity was
II-6

Fenofibrate

mentioned on the JPN label.

II-7

Furosemide

Furosemide was selected from the list of positive chemicals used in the 3T3 NRU-PT

39
45

validation study. Human data for this chemical was described as positive in the article on
the 3T3 NRU-PT validation study [Spielmann et al, 1998a].

Although the 2011 USlabel did mention photosensitivity, 2012 US label did not. Photosensitivity wasmentioned on the JPN label.
Ketoprofen was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on
the 3T3 NRU-PT validation study [Spielmann et al, 1998a].
II-8

Ketoprofen

Photosensitivity wasmentioned on both the US and the JPN label.
6-Methylcoumarine was selected from the list of positive chemicals used in the 3T3
NRU-PT validation study. Human data for this chemical was described as positive in the

II-9

6-Methylcoumarine

article on the 3T3 NRU-PT validation study [Spielmann et al, 1998a].
8-MOP was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on
the 3T3 NRU-PT validation study [Spielmann et al, 1994a].

II-10

8-MOP

Photosensitivity wasmentioned on both the US and the JPN label.
Nalidixic acid was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on
the 3T3 NRU-PT validation study [Spielmann et al, 1998a].
Nalidixic acid (II-11) and nalidixic acid (Na salt) (II-12) were tested both as a free form
and a salt in order to test whether the aqueous solubility of chemicals might limit thepredictive power of the ROS assay. Photosensitivity was mentioned on both the US and

II-11

Nalidixic acid

the JPN label.
Nalidixic acid (Na salt) was selected from the list of positive chemicals used in the 3T3
NRU-PT validation study. Human data for this chemical was described as positive in thearticle on the 3T3 NRU-PT validation study [Spielmann et al, 1998a].
Nalidixic acid (II-11) and nalidixic acid (Na salt) (II-12) were tested both as a free form
and a salt in order to test whether the aqueous solubility of chemicals might limit thepredictive power of the ROS assay. Photosensitivity was mentioned on both the US and

II-12

Nalidixic acid (Na salt)

the JPN label.
Norfloxacin was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on
the 3T3 NRU-PT validation study [Spielmann et al, 1998a].

II-13

Norfloxacin

Photosensitivity wasmentioned on both the US and the JPN label.
Ofloxacin was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on

II-14

Ofloxacin

the 3T3 NRU-PT validation study [Spielmann et al, 1998a].

40
46

Photosensitivity wasmentioned on both the US and the JPN label.
Piroxicam was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on
the 3T3 NRU-PT validation study [Spielmann et al, 1994a].
II-15

Piroxicam

Photosensitivity wasmentioned on both the US and the JPN label.
Promethazine HCl was selected from the list of positive chemicals used in the 3T3
NRU-PT validation study. Human data for this chemical was described as positive in thearticle on the 3T3 NRU-PT validation study [Spielmann et al, 1994a].

II-16

Promethazine HCl

Photosensitivity

was mentioned on both the US and the JPN label.
Rosiglitazone was selected because 3T3 NRU-PT was positive, but high quality human

II-17

Rosiglitazone

data regarding phototoxicity was not available.
Tetracycline was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on
the 3T3 NRU-PT validation study [Spielmann et al, 1994a].

II-18

Tetracycline

Photosensitivity wasmentioned on both the US and the JPN label.

Phototoxic non-drug chemicals
Anthracene was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on
II-19

Anthracene

the 3T3 NRU-PT validation study [Spielmann et al, 1998a].
Avobenzone was reported with photoallergy negative by a photopatch test in the article
by Szczurko C et al. [1994] and Trevisi P et al. [1994], but was reported to induce
photoallergic reactions in the article by Schauder, S. et al. [1997].

Therefore,

avobenzone was classified as a phototoxic chemical because we were not able to
II-20

Avobenzone

completely judge that avobenzone was a non-phototoxic chemical.
Bithionol was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on

II-21

Bithionolthe 3T3 NRU-PT validation study [Spielmann et al, 1994a].
Hexachlorophene was selected from the list of positive chemicals used in the 3T3
NRU-PT validation study. Human data for this chemical was described as positive in thearticle on the 3T3 NRU-PT validation study [Masuda et al., 1971 and Spielmann et al,

II-22

Hexachlorophene

1998a].
Rose bengal was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on

II-23

Rose bengal

the 3T3 NRU-PT validation study [Spielmann et al, 1998a].

Non-phototoxic drugs

41
47

Aspirin was selected based on negative results of the 3T3 NRU-PT per the article byOnoue et al. [2010].
II-24

AspirinPhotosensitivity was no䡐 mentioned on either the US or the JPN

label.
Benzocaine was selected based on negative results of the 3T3 NRU-PT per the article byOnoue et al. [2010].

II-25

Benzocaine

Photosensitivity was no䡐 mentioned on either the US or the JPN

label.
Erythromycin was selected based on negative results of the 3T3 NRU-PT per the article
by Onoue et al. [2010].

II-26

Erythromycin

Photosensitivity was no䡐 mentioned on either the US or the

JPN label.
Penicillin G was selected from the list of negative chemicals used in the 3T3 NRU-PT
validation study [Spielmann et al, 1994a].

II-27

Penicillin G

Photosensitivity was no䡐 mentioned oneither the US or the JPN label.
Phenytoin was selected based on negative results of the 3T3 NRU-PT per the article byOnoue et al. [2010].

II-28

Phenytoin

Photosensitivity was no䡐 mentioned on either the US or the JPN

label.

Non-phototoxic non-drug chemicals
Bumetrizole was selected from UV absorbers. We thought that human data of this UV
absorber would be provided.

Human data for this chemical, however, was notavailable. Therefore after confirming negative result in 3T3 NRU-PT, this chemical
II-29

Bumetrizole

was classified as a non-phototoxic chemical.
Chemical No. II-30 was registered at first as benzylindene camphor sulphonic acid. This
was a UV absorber used in the 3T3 NRU-PT validation study. However, we carried out
the Phase 2 study without noticing a transcription error of the CAS number on the final
chemical list. Because we did not notice this mistake until after the Phase 2 study, thischemical was reclassified as a non-phototoxic chemical after confirming negative results

II-30

Camphor sulfonic acidin 3T3 NRU-PT and low molar extinction coefficient (MEC).
Chlorhexidine was selected from the list of negative chemicals used in the 3T3 NRU-PT
validation study [Spielmann et al, 1994a and 1998a]. Negative result in human was

II-31

Chlorhexidine

described in the article on the 3T3 NRU-PT validation study [Spielmann et al, 1998a].
We originally intended to use cinnamic aldehyde, an aromatic ingredient used in
cinnamon, as Chemical No. II-32, because this chemical used in the 3T3 NRU-PT
validation study. However, we inadvertently added cinnamic acid instead of cinnamic
aldehyde to the list of chemicals.

Cinnamic acid is known to form a dimer by lightirradiation when in the solid state.

The difference between cinnamic aldehyde andcinnamic acid is that the former has a side chain of aldehyde and the later one of
II-32

Cinnamic acidcarboxyl.

In addition, we carried out 3T3NRU-PT for cinnamic acid and the result was

42
48

negative, just like cinnamic aldehyde.

Therefore, we concluded that either chemical

was suitable for the ROS assay validation study, because cinnamic acid resembles
cinnamic aldehyde structurally, and the result of 3T3 NRU-PT testing was the same.
Drometrizole was selected from UV absorbers. We thought that human data of this UV
absorber would be provided.

Human data for this chemical, however, was notavailable. Therefore after confirming negative result in 3T3 NRU-PT, this chemical
II-33

Drometrizole

was classified as a non-phototoxic chemical.
L-Histidine was selected from the list of negative chemicals used in the 3T3 NRU-PT

II-34

L-Histidine

validation study [Spielmann et al, 1994a].
Methylbenzylidene camphor was selected from UV absorbers.

This was in the list ofnegative chemicals in the 3T3 NRU-PT validation study and Human data for thisII-35

Methylbenzylidene

chemical was described as negative in the article on the 3T3 NRU-PT validation study

camphor[Spielmann et al, 1998b].
Octrizole was selected from UV absorbers. We thought that human data of this UV
absorber would be provided.

Human data for this chemical, however, was notavailable. Therefore after confirming negative result in 3T3 NRU-PT, this chemical
II-36

Octrizole

was classified as a non-phototoxic chemical.
Chemical No. II-37 was registered at first as octyl methoxycinnamate. This was UV
absorber used in the 3T3 NRU-PT validation study. However, we have carried out thePhase 2 study without noticing a transcribing error of the CAS number when we madethe final chemical list. Because we noticed to take it wrong after the Phase 2 study, thischemical was classified as non-phototoxic chemical again after confirming negative

II-37

Octyl methacrylate

result in 3T3 NRU-PT and low molar extinction coefficient (MEC).
Octyl methoxycinnamate was selected from UV absorbers.

This was in the list ofnegative chemicals in the 3T3 NRU-PT validation study and Human data for thischemical was described as negative in the article on the 3T3 NRU-PT validation study
II-38

Octyl methoxycinnamate[Spielmann et al, 1998b].
Octyl salicylate was selected from UV absorbers.

This was in the list of negative

chemicals in the 3T3 NRU-PT validation study and Human data for this chemical was
described as negative in the article on the 3T3 NRU-PT validation study [Spielmann etII-39

Octyl salicylateal, 1998b].
PABA was selected from the list of negative chemicals used in the 3T3 NRU-PT
validation study [Spielmann et al, 1994a and 1998a].

II-40

PABA

Negative result in animals was

described in the article on the 3T3 NRU-PT validation study [Spielmann et al, 1998a].
SDS was selected from the list of negative chemicals used in the 3T3 NRU-PT validation

II-41

SDS

study [Spielmann et al, 1994a].

43
49

UV-571 was selected from UV absorbers.
absorber would be provided.

We thought that human data of this UV

Human data for this chemical, however, was notavailable. Therefore after confirming negative result in 3T3 NRU-PT, this chemical
II-42

UV-571was classified as a non-phototoxic chemical.

Positive/Negative controls
Quinine HCl was used as positive control according to the method of Onoue et al.
[2008a].
PC

Quinine HCl

Quinine HCl was classified as a phototoxic chemical for human per the article

by Ljunggren et al [1986].

Photosensitivity was mentioned on the US label.

Sulizobenzone was used as negative control according to the method of Onoue et al.
[2008a].

Sulisobenzone was classified as a non-phototoxic chemical, because Humandata for this chemical was described as negative in the article on the 3T3 NRU-PT
NC

Sulisobenzone

validation study [Spielmann et al, 1998b].

44
50

Appendix 5 Test chemicals for Phase 2 study and code listin vivo

UV/vis absorptionb)

No.

Chemical name

CAS No.a)Laboratory

3T3
MEC

Ȝmax

(L/mol/cm)

(nm)

NRU

Animal

Human

1

2

3

Phototoxic drugs
II-1

Acridine

260-94-6

2773

354

+ 1)

+ 1)

+ 1)

C-130

B-090

A-005

II-2

Acridine HCl

17784-47-3

2635

354

+ 1)

+ 1)

+ 1)

C-126

B-086

A-001

II-3

Amiodarone HCl

19774-82-4

5400

290 c)

+ 2)

+ 2)

+ 2)

C-127

B-087

A-002

2)

2)

2)

C-106

B-066

A-026

II-4

Chlorpromazine HCl

69-09-0

1746

304

+

+

+

II-5

Doxycycline HCl

10592-13-93715

290 c)

+ 2)

+ 2)

+ 2)

C-116

B-076

A-036

II-6

Fenofibrate

49562-28-93514

290 c)

+ 1)

NA

+ 1)

C-139

B-054

A-014

+/- 1,3,4)

NA

+

1)

C-141

B-056

A-016

II-7

Furosemide

54-31-92650

290 c)

II-8

Ketoprofen

22071-15-4

2092

290 c)

+ 1)

- 1)

+ 1)

C-128

B-088

A-003

II-9

6-Methylcoumarine

92-48-83219

290 c)

+ 1)

+ 1)

+ 1)

C-113

B-073

A-033

II-10

8-MOP

298-81-7

3631

300

+ 2)

+ 2)

+ 2)

C-131

B-091

A-006

II-11

Nalidixic acid389-08-2

3192

331

+ 1)

+ 1)

+ 1)

C-137

B-052

A-012

II-12

Nalidixic acid (Na salt)

3374-05-8

3019

333

+ 1)

+ 1)

+ 1)

C-134

B-094

A-009

II-13

Norfloxacin70458-96-7

3562

323

+ 3)

+ 1)

+ 1)

C-110

B-070

A-030

II-14

Ofloxacin

82419-36-1

8443

290 c)

+ 1)

+ 1)

+ 1)

C-112

B-072

A-032

II-15

Piroxicam

36322-90-4

3304

352

- 2)

- 2)

+ 2)

C-135

B-095

A-010

II-16

Promethazine HCl

58-33-3

1558

297

+ 2)

NA

+ 2)

C-101

B-061

A-021

II-17

Rosiglitazone

122320-73-4

1765

311

+ d)

NA

NA

C-117

B-077

A-037

II-18

Tetracycline

60-54-83842

290 c)

+ 2)

+ 2)

+ 2)

C-102

B-062

A-022

+ 1)

+ 1)

C-121

B-081

A-041

7)

C-109

B-069

A-029

Phototoxic non-drug chemicals
II-19

Anthracene120-12-7

2315

355

+ 1)

II-20

Avobenzone

70356-09-1

7686

354

+

II-21

Bithionol

97-18-7

2462

321

+ 2)

+ 2)

+ 2)

C-115

B-075

A-035

1)

1)

+ 1,8)

C-107

B-067

A-027

5)

-

-

6)

+

II-22

Hexachlorophene70-30-4

2431

300

-

II-23

Rose bengal

632-69-9

19269

549

+ 1)

- 1)

+ 1)

C-104

B-064

A-024

Non-phototoxic drugs
II-24

Aspirin

50-78-2

80

290 c)

- 3)

NA

NA

C-140

B-055

A-015

II-25

Benzocaine

94-09-74273

290 c)

- 3)

NA

NA

C-114

B-074

A-034

-

3)

NA

NA

C-119

B-079

A-039

II-26

Erythromycin

114-07-80

290 c)

II-27

Penicillin G

113-98-40

290 c)

- 2)

NA

NA

C-118

B-078

A-038

0

290 c)

3)

NA

NA

C-145

B-060

A-020

II-28

Phenytoin

57-41-0-45
51

Appendix 5 Test chemicals for Phase 2 study and code list
(continued)
in vivo

UV/vis absorptionb)

No.

Chemical name

CAS No.a)Laboratory

3T3
MEC

Ȝmax

(L/mol/cm)(nm)

3896-11-5

3873

3144-16-9

NRU

Animal

Human

1

2

3

306

- d)

NA

NA

C-138

B-053

A-013

0

290 c)

- d)

NA

NA

C-132

B-092

A-007

Non-phototoxic non-drug chemicals
II-29
II-30

Bumetrizole
Camphor sulfonic
acid

II-31

Chlorhexidine

55-56-1

1338

290 c)

- 1)

NA

- 1)

C-144

B-059

A-019

II-32

Cinnamic acid140-10-3

3373

290 c)

- d)

NA

NA

C-123

B-083

A-043

II-33

Drometrizole

2440-22-4

3946

295

- d)

NA

NA

C-129

B-089

A-004

II-34

L-Histidine

71-00-1

0

290 c)

- 2)

NA

NA

C-111

B-071

A-031

36861-47-9

9200

304

- 9)

- 9)

- 9)

C-136

B-051

A-011

II-35

Methylbenzylidene
camphor

II-36

Octrizole3147-75-9

3958

296

- d)

NA

NA

C-133

B-093

A-008

II-37

Octyl methacrylate

688-84-6

0

290 c)

-

d)

NA

NA

C-105

B-065

A-025

5466-77-3

3000

290 c)

- 9)

- 9)

- 9)

C-142

B-057

A-017

II-38

Octyl
methoxycinnamate

II-39

Octyl salicylate

118-60-5

1500

290 c)

- 9)

- 9)

- 9)

C-120

B-080

A-040

II-40

PABA

150-13-02404

290 c)

- 2)

- 1)

NA

C-124

B-084

A-044

c)

2)

NA

NA

C-125

B-085

A-045

II-41

SDS

151-21-3

0

290

-

II-42

UV-571125304-04-3

1900

290 c)

- d)

NA

NA

C-122

B-082

A-042

Positive/Negative controls
PC

Quinine HCl

6119-47-7

1938

330

+ 3)

+ 10)

+ 11)

PC

PC

PC

NC

Sulisobenzone

4065-45-6

3519

290 c)

- 3)

NA

- 9)

NC

NC

NC

8-MOP: 8-methoxy psoralen, PABA: p-aminobenzoic acid, SDS: sodium dodecyl sulfate+ : Positive, - : Negative, +/- : Equivocal, NA : Not available, PC : Positive control, NC : Negative controla) CAS No.: Chemical abstracts service registry number, b) The UV/vis absorbance (290-700 nm) of most chemicals
was measured in 20 mM phosphate buffer (pH 7.4). However, the UV/vis absorbance of chemical Nos. II-19, II-20,
II-29, II-33 and II-36 were measured in methanol, because these chemicals were not solved in 20 mM phosphatebuffer (pH 7.4). In the each case, test chemicals were dissolved in DMSO at 10 mM and diluted with 20 mM
phosphate buffer (pH 7.4) or methanol. Final concentration of DMSO was unified to 0.5%. c) Ȝmax (nm) was a
wavelength at which the UV/vis absorbance shows a peak between 290 and 700 nm. Ȝmax (nm) was indicated as 290
nm in the case where the peak absorption is located below 290 nm and the maximum absorption is at 290 nm. d) In
vitro phototoxicity was assessed by the 3T3 NRU PT in the participating laboratories, according to the OECD 432
guideline.
1) Spielmann et al., 1998a, 2) Spielmann et al., 1994a, 3) Onoue et al., 2010, 4) Peters et al., 2002, 5) Gaspar et al.,2012, 6) ZEBET in house data, 7) Schauder et al., 1997, 8) Masuda et al., 1971, 9) Spielmann et al., 1998b, 10)Spielmann et al., 1994b, 11) Ljunggren et al., 1986

46
52