༤
༤ኈㄽᩥ
HepaRG ⣽⬊ࢆ⏝࠸ࡓ
௦ㅰⓗάᛶࡼࡿ⫢㞀ᐖࡢホ౯
ᗈᓥᏛᏛ㝔་ṑ⸆ಖᏛ◊✲⛉
་ṑ⸆Ꮫᑓᨷ
⏕యᶵ⬟ศᏊືែᏛ◊✲ᐊ
ᖹᡂ
25 ᖺᗘධᏛ ᶓᒣ 㞝୍
␎
␎ྕ୍ぴ
CYP: cytochrome P450
UGT: uridine 5’-diphospho-glucuronosyltranseferase
SULT: sulfotransferase
GLP: good laboratory practice
ATP: adenosine triphosphate
LDH: lactate dehydrogenase
DMSO: dimethyl sulfoxide
AFB1: aflatoxin B1 CPA: cyclophosphamide APAP: acetaminophen TAM: tamoxifen TGZ: troglitazone ABT: 1-aminobenzotriazole GSH: glutathione
BSO: L-buthionine-(S, R)-sulfoximine
NAPQI: N-acethyl-p-benzoquinone imine
SAM: salicylamide
GAPDH: glyceraldehyde 3-phosphate dehydrogenase
┠ḟ
࠙␎ㄒ୍ぴࠚ 1. ➨ 1 ❶ ᗎㄽ ... 1 2. ➨ 2 ❶ ⫢⣽⬊ホ౯⣔ࡢ⸆≀௦ㅰ㓝⣲άᛶホ౯ ... 4 2.1 ➨1 ⠇ ⥴ゝ ... 4 2.2 ➨2 ⠇ HepaRG ⣽⬊ࡢ⸆≀௦ㅰ㓝⣲άᛶホ౯ ... 4 2.2.1 ➨ 1 㡯 HepaRG ⣽⬊ࡢศᇵ㣴᮲௳ ... 4 2.2.2 ➨ 2 㡯 ศࡋࡓ HepaRG ⣽⬊ࡢ⸆≀௦ㅰ㓝⣲άᛶホ౯ ... 7 2.3 ➨3 ⠇ ࣄࢺ⫢ࡀࢇ⏤᮶⣽⬊ HepG2 ⣽⬊ࡢ⸆≀௦ㅰ㓝⣲άᛶホ౯ ... 7 2.4 ➨4 ⠇ ࣄࢺ⤖⫢⣽⬊ࡢ⸆≀௦ㅰ㓝⣲άᛶホ౯ ... 8 2.5 ➨5 ⠇ ᑠᣓ ... 9 3. ➨ 3 ❶ ⫢ẘᛶ≀㉁ࢆ⏝࠸ࡓ⣽⬊ẘᛶホ౯ ...11 3.1 ➨1 ⠇ ⥴ゝ ...11 3.2 ➨2 ⠇ ࣄࢺ⤖⫢⣽⬊ࢆ⏝࠸ࡓ⣽⬊ẘᛶホ౯ ... 12 3.3 ➨3 ⠇ HepaRG ⣽⬊ࢆ⏝࠸ࡓ⣽⬊ẘᛶホ౯ ... 13 3.4 ➨4 ⠇ ᑠᣓ ... 14 4. ➨ 4 ❶ ⫢ẘᛶ≀㉁ࢆ⏝࠸ࡓ⥙⨶ⓗ㑇ఏᏊⓎ⌧ኚືゎᯒ ... 16 4.1 ➨1 ⠇ ⥴ゝ ... 16 4.2 ➨2 ⠇ HepaRG ⣽⬊ཬࡧ HepG2 ⣽⬊ࢆ⏝࠸ࡓ㑇ఏᏊⓎ⌧ゎᯒ ... 17 4.3 ➨3 ⠇ ᑠᣓ ... 20 5. ➨ 5 ❶ ⫢ẘᛶ≀㉁ࡢ௦ㅰ≀ࡼࡿẘᛶⓎ⌧ホ౯ ... 21 5.1 ➨1 ⠇ ⥴ゝ ... 21 5.2 ➨2 ⠇ ⣽⬊ẘᛶⓎ⌧ᑐࡍࡿ CYP ࡼࡿ௦ㅰࡢᙳ㡪 ... 22 5.2.1 ➨ 1 㡯 ࣄࢺ⤖⫢⣽⬊ࢆ⏝࠸ࡓ⣽⬊ẘᛶホ౯ ... 22 5.2.2 ➨ 2 㡯 HepaRG ⣽⬊ࢆ⏝࠸ࡓ⣽⬊ẘᛶホ౯ ... 25 5.3 ➨3 ⠇ ⣽⬊ẘᛶᑐࡍࡿ⏕యෆᢠ㓟≀㉁ GSH ࡢᙳ㡪 ... 30 5.4 ➨4 ⠇ ࢭࢺ࣑ࣀࣇ࢙ࣥࡢẘᛶⓎ⌧ᑐࡍࡿ௦ㅰ≀ࡢᙳ㡪ࡢホ౯ ... 33 5.5 ➨5 ⠇ ᑠᣓ ... 37 6. ➨ 6 ❶ ⪃ᐹ⥲ᣓ ... 38 ࠙ᐇ㦂ࡢ㒊ࠚ- - - 42 ࠙ᘬ⏝ᩥ⊩ࠚ- - - 51 ࠙ᮏㄽᩥࡢᇶ♏࡞ࡿཎⴭࠚ- - - 58 ࠙ㅰ㎡ࠚ - - - 591.
➨
1 ❶ ᗎㄽ
㏆ᖺ㸪᪂つ⸆ᶆⓗศᏊࡢᯤῬࡼࡾ㸪పศᏊྜ≀ࡢ⸆ࡀᅔ㞴࡞ࡗ࡚ࡁ࡚࠸ࡿ⪃࠼ࡽ ࢀ࡚࠾ࡾ㸪ࡑࡢࡓࡵ㸪㝈ࡽࢀࡓೃ⿵ྜ≀ࢆ☜ᗘ㧗ࡃ⸆࣭㛤Ⓨࡍࡿࡇࡀồࡵࡽࢀ࡚࠸ࡿ㸬་ ⸆ရࡢ㛤Ⓨࢫࢸ࣮ࢪ࡛ࡢ㛤Ⓨ୰Ṇࡢせᅉࡢ࠺ࡕ㸪Ᏻᛶ㠃࡛ࡢၥ㢟ࡀཎᅉ࡞ࡿሙྜࡢ༨ࡵࡿ ྜࡀ㧗࠸㸦Kola and Landis 2004; Hornberg et al., 2014㸧㸬Waring ࡽࡢሗ࿌ࡼࡿᾏእᡭ〇⸆࣓
࣮࣮࢝ࢆᑐ㇟ࡋࡓゎᯒ⤖ᯝ࡛㸪⤒ཱྀపศᏊ་⸆ရࡢ㛤Ⓨೃ⿵ရ812 ྜ≀ࡢ୰࡛㸪ୖᕷࡲ࡛⮳ ࡽࡎ㛤Ⓨ୰Ṇ࡞ࡗࡓ605 ྜ≀ࡢෆ㸪240 ྜ≀㸦⣙ 40%㸧ࡀ㠀⮫ᗋヨ㦂࡛ࡢẘᛶࡀཎᅉ࡛ ࠶ࡗࡓࡢሗ࿌ࡶ࠶ࡿ㸦Waring et al., 2015㸧㸬⸆ຠ㠃࡛᭷ᮃ࡞࠸ࡃࡘࡢ㛤Ⓨೃ⿵ྜ≀ࡢෆ㸪Ᏻ ᛶ࣭ẘᛶ⌮⏤ࡼࡾ㛤Ⓨ୰Ṇ࡞ࡿࣜࢫࢡࡢᑡ࡞࠸ྜ≀ࢆ㑅ฟࡍࡿࡓࡵ㸪⸆ࡢึᮇẁ㝵 ࡽẘᛶࡢࣜࢫࢡࢆண 㸪ᅇ㑊ࡍࡿࡓࡵࡢホ౯⣔ࡀྍḞ࡛࠶ࡿ㸬 Ᏻᛶࡀၥ㢟࡞ࡾ་⸆ရ㛤Ⓨࡀ୰Ṇ࡞ࡿࢣ࣮ࢫ࠾࠸࡚㸪᭱ࡶせ࡞ཎᅉࡢࡦࡘ⫢㞀 ᐖࡀᣲࡆࡽࢀࡿ㸦Hornberg et al., 2014㸧㸬⸆ㄏⓎᛶ⫢㞀ᐖᑐࡍࡿ◊✲ࡣẚ㍑ⓗ┒ࢇ⾜ࢃࢀ࡚ ࠸ࡿࡀ㸪Idiosyncratic㸦≉␗య㉁ᛶ㸧࡞⫢㞀ᐖ࡞㸪Ⓨ⌧࣓࢝ࢽࢬ࣒ࡀ༑ศゎ᫂ࡉࢀ࡚࠸࡞࠸ࡶࡢ ࡶ࠶ࡾ㸪ᮍࡔ༑ศࡣゝ࠼࡞࠸㸬 ⸆ࡢࣉࣟࢭࢫࡣ୍⯡ⓗࠗࢱ࣮ࢤࢵࢺⓎ᥀࣭ࣄࢵࢺྜ≀ࡢ᥈⣴࣭ྜ≀ࡢ᭱㐺࣭㛤Ⓨೃ ⿵ရࡢ㑅ฟ࣭⏦ㄳᚲせ࡞㠀⮫ᗋࢹ࣮ࢱࡢྲྀᚓ࠘࠸࠺ὶࢀ༊ษࡽࢀࡿ㸬ࡑࡢ୰࡛㸪Ᏻᛶホ ౯㛵ࡋ࡚ࡣ㸪ྜ≀᭱㐺࣭㑅ᢤࡢ㐣⛬࡛ᐇࡍࡿ᥈⣴ⓗ࡞ẘᛶヨ㦂㸪㛤Ⓨೃ⿵ရࡘ࠸࡚ᐇ ࡍࡿࣞࢠࣗࣛࢺ࣮ࣜᑐᛂࡋࡓGLP ẘᛶヨ㦂࠸࠺㐣⛬࡛⾜ࢃࢀࡿ㸦Fig. 1㸧㸬᥈⣴ⓗ࡞ẘᛶヨ 㦂࡛ࡣin vitro ཬࡧ in vivo ࡢヨ㦂⣔ࢆ⏝࠸࡚㸪㛤Ⓨೃ⿵㑅ฟࡍࡿྜ≀ࡢẘᛶ࣏ࢸࣥࢩࣕࣝࡢ ᢕᥱ࠾ࡼࡧࡼࡾẘᛶᠱᛕࡢ࡞࠸ೃ⿵ရࢆ㑅ฟࡍࡿࡓࡵࡢࢫࢡ࣮ࣜࢽࣥࢢࢆ⾜࠸㸪GLP ẘᛶヨ㦂࡛ ࡣ㸪㛤Ⓨೃ⿵ရࡢẘᛶࣉࣟࣇ࣭ࣝ⸆ຠᑐࡍࡿᏳᇦࡢ⟬ฟࢆ⾜࠺ࡓࡵࡢࢹ࣮ࢱࢆྲྀᚓࡋ㸪 ⮫ᗋ㛤Ⓨࢆ㐍ࡵ࡚࠸ࡃὶࢀ࡞ࡿ㸬㛤Ⓨೃ⿵ရࡀᑠື≀㸦࣐࢘ࢫ㸪ࣛࢵࢺ㸧㸪ື≀㸦ࢾ㸪ࢧ ࣝ㸧࡞ࢆ⏝࠸ࡓ㠀⮫ᗋࡢẘᛶヨ㦂࡛⫢㞀ᐖᛶࢆ♧ࡋࡓሙྜ㸪ࡑࡢẘᛶⓎ⌧࣓࢝ࢽࢬ࣒ࡢゎᯒ࣭ ࣄࢺࡢእᤄᛶࡢ᳨ドࢆ⾜࠸㸪࣓࢝ࢽࢬ࣒ᛂࡌࡓ⫢㞀ᐖᛶᅇ㑊ࡢࡓࡵࡢࢫࢡ࣮ࣜࢽࣥࢢ⣔ࡢᵓ ⠏㸪ẘᛶᅇ㑊ྜ≀ࡢ㑅ฟ࠸࠺ὶࢀ࡛㸪⸆◊✲࠾ࡅࡿ⫢ẘᛶࡢᑐᛂࡀ⾜ࢃࢀࡿࡢࡀ୍⯡ ⓗ࡛࠶ࡿ㸬ࡇࡢẘᛶⓎ⌧࣓࢝ࢽࢬ࣒ࡢゎᯒࡸ㸪ࢫࢡ࣮ࣜࢽࣥࢢ⣔ࡢᵓ⠏࠾࠸࡚㸪ࣄࢺࡢ⫢㞀 ᐖᛶࢆホ౯ࡍࡿࡓࡵࡢࣄࢺ⫢⣽⬊ࣔࢹࣝࡀᚲせ࡞ࡿ㸬 Target Hit Identification Lead optimization Hit to lead
Drug Discovery
Candidate selection Preclinical development Clinical developmentDrug development
ࣄࢺ⫢⮚ᇵ㣴⣽⬊⣔ࡢࢦ࣮ࣝࢹࣥࢫࢱࣥࢲ࣮ࢻࡋ࡚⏝ࡉࢀࡿࡢࡀ㸪ࣄࢺ⫢⣽⬊࡛࠶ࡿ㸬ࣄ ࢺ⫢⣽⬊ࡢ୰࡛ࡶࣄࢺ᪂㩭⫢⣽⬊㸪ࣄࢺ⤖⫢⣽⬊࡞ࡀ࠶ࡿ.᪂㩭⫢⣽⬊ࡣධᡭࡀᐜ࡛᫆ࡣ࡞ࡃ ⸆◊✲࡛ࡢ⏝ࡣ㞴ࡋ࠸ࡀ㸪⤖⫢⣽⬊ࡣẚ㍑ⓗᐜ᫆ධᡭ࡛ࡁ㸪⸆࠾ࡅࡿ⸆≀ືែ◊✲㸪 ẘᛶ◊✲⏝ࡉࢀࡿࡇࡀከ࠸㸬ࣄࢺ⤖⫢⣽⬊ࡣࡑࡢ⤖ಖᏑᢏ⾡ࡢⓎᒎక࠸㸪⸆≀௦ㅰ άᛶ࡞ࡢᶵ⬟ࢆ⥔ᣢࡋࡓ≧ែ࡛ධᡭࡍࡿࡇࡀྍ⬟࡞ࡾ㸪௦ㅰάᛶࡢ㧗࠸ࣄࢺ⫢⣽⬊ࢆヨ㦂 ◊✲⏝࡛ࡁࡿࡼ࠺࡞ࡗࡓ㸬ࡋࡋ࡞ࡀࡽ㸪ࣄࢺ⤖⫢⣽⬊ࡣࢻࢼ࣮ࡢಶయ⏤᮶ࡍࡿ⸆≀ ௦ㅰ㓝⣲ࡢࣟࢵࢺ㛫ᕪࡀࡁ࠸࠸࠺ḞⅬࡀ࠶ࡿ㸬ࡲࡓࣟࢵࢺࡼࡗ࡚ࡣ୍㒊ࡢ௦ㅰ㓝⣲άᛶࡀ ప࠸ࣟࢵࢺࡀᏑᅾࡍࡿሙྜ࡞ࡀ࠶ࡾ㸪⸆◊✲㸪≉Ᏻᐃ࡞ホ౯⣔ࡀᚲせ࡞ึᮇࡢࢫࢡ࣮ࣜࢽ ࣥࢢ࡞࡛ࡣ⏝ࡋ࡙ࡽ࠸࠸࠺ḞⅬࡀ࠶ࡿ㸬ࡉࡽ㸪✀┤ᚋࡸᠱ⃮≧ែ࡛ࡢ⏝࡛ࡣ⸆≀௦ ㅰ⬟ࡀ㧗࠸ࡀ㸪ẘᛶホ౯࡞ࡢ༢ᒙᇵ㣴࡞ࡢ᥋╔≧ែ࡛ヨ㦂ࢆ⾜࠺ሙྜ㸪୍ᬌ᥋╔ᚋ㸪௦ㅰά ᛶࡀⴭࡋࡃపୗࡋ࡚ࡋࡲ࠺࡞ࡢㄢ㢟ࡶ࠶ࡿ㸬௦ㅰάᛶࡀ㧗ࡃ㸪ศᏊ✀ࡼࡾάᛶ್Ḟᦆࡀ࡞ ࠸㸪௦ㅰࣉࣟࣇࣝࡢⰋዲ࡞ࣟࢵࢺࡶᏑᅾࡍࡿࡀ㸪ࡑࡢࡼ࠺࡞ࣟࢵࢺࡣ୍ᐃࡢ⏝⪅ࣟࢵࢺ ࡈໟᣓ㉎ධࡉࢀධᡭ࡛ࡁ࡞࠸࡞㸪᭷ᮃ࡞ࣟࢵࢺࡢᏳᐃⓗ࡞౪⤥ࡶ㞴ࡋ࠸㸬ࡲࡓ㸪ࣄࢺ⤖⫢ ⣽⬊ࡣ㸪ẚ㍑ⓗ㧗౯࡛࠶ࡾ㸪ึᮇࡢࢫࢡ࣮ࣜࢽࣥࢢ࡛⣽⬊ࢆ㔞⏝ࡍࡿ⸆ࡢึᮇẁ㝵࡛ࡣ ⏝ࡀ㞴ࡋ࠸࠸࠺ḞⅬࡀ࠶ࡿ㸬୍᪉㸪ᶵ⬟࡞ࡀᆒ㉁࡛ࣟࢵࢺ㛫ᕪ࡞ࡀᑡ࡞࠸⣽⬊ࡋ࡚㸪 ᰴࡉࢀࡓࢭࣝࣛࣥ࡞ࡀᣲࡆࡽࢀ㸪⫢⣽⬊ࡘ࠸࡚ࡶࣄࢺ⫢⭘⒆⏤᮶⣽⬊ᰴࡢHepG2 ⣽⬊࡞ ࡀỗ⏝ᛶ㧗ࡃ⏝ࡉࢀ࡚࠸ࡿ㸬ࡇࢀࡽࡢ⣽⬊ࡣẚ㍑ⓗᇵ㣴ࡶᐜ࡛᫆㸪ᶵ⬟࡞ࡶᏳᐃࡋ࡚࠸ࡿ ࡓࡵ㸪ึᮇࡢࢫࢡ࣮ࣜࢽࣥࢢ⏝ࡋࡸࡍ࠸࠸࠺Ⅼࡀ࠶ࡿࡀ㸪ከࡃࡢ⸆≀௦ㅰ㓝⣲ࡢⓎ⌧ࡀ ࣄࢺ⫢⣽⬊ẚ㍑ࡋ㠀ᖖప࠸ࡓࡵ㸪ࣄࢺࡢ⫢⮚ࡢᶵ⬟ࡢ≉ᚩࡢ୍ࡘ࡛࠶ࡿ⸆≀௦ㅰ㓝⣲ࡢᙳ㡪 ࢆ༑ศࡣホ౯࡛ࡁ࡞࠸㸬ẘᛶホ౯ࡢほⅬ࡛ࡶ㸪௦ㅰࢆࡋࡓྜ≀ࡢᙳ㡪ࡸ㸪௦ㅰ≀ࡢẘᛶࢆ 㐣ᑠホ౯ࡍࡿྍ⬟ᛶࡀ࠶ࡿ㸬ࡇࡢࡼ࠺㸪⸆◊✲࠾࠸࡚㸪ࣄࢺࡢ⸆≀௦ㅰᶵ⬟ࢆᏳᐃⓗ᭷ ࡋ㸪ẘᛶࡢࢫࢡ࣮ࣜࢽࣥࢢࡸ࣓࢝ࢽࢬ࣒ゎᯒ⏝࡛ࡁࡿ⫢⣽⬊ࣔࢹࣿࡀᮃࡲࢀࡿࡀ㸪ࡑࢀࡒࢀ ࡢ⫢⣽⬊ࣔࢹࣝࡣㄢ㢟ࡀ࠶ࡿ㸬
HepaRG ⣽⬊ࡣ 2002 ᖺ Gripon ࡽࡼࡾᶞ❧࣭ሗ࿌ࡉࢀࡓࣄࢺ⫢⭘⒆⏤᮶ࡢ⣽⬊ᰴ࡛࠶ࡿࡀ 㸦Gripon et al., 2002㸧㸪DMSO ཬࡧ hydrocortisone ࡞ࡢ௦⾲ⓗ࡞ศㄏᑟࡀྵࡲࢀࡿ≉Ṧ࡞ᇵ ᆅࢆ⏝࠸ࡓᇵ㣴᮲௳୍࡛ᐃᮇ㛫ᇵ㣴ࡋศࡉࡏࡿ㸪⫢⣽⬊ᵝࡢᙧែࢆ♧ࡍ⣽⬊⫹⟶ୖ⓶ᵝࡢ
ᙧែࢆ♧ࡍ 2 ࡘࡢᙧែࢆࡶࡘ⣽⬊ࡢඹᇵ㣴⣔ࡢ≧ែࢆ♧ࡍࡼ࠺࡞ࡿ㸬ࡇࡢࡼ࠺ศࡉࢀࡓ
HepaRG ⣽⬊ࡣᙧែࡢࡳ࡞ࡽࡎ㸪⫢⮚≉␗ⓗ࡞ albumin㸪aldolase B ࡞ࡢⓎ⌧ຍ࠼㸪ẟ⣽⫹ ⟶ࡢⓎ⌧ࡀㄆࡵࡽࢀࡿ࡞ࡢ㸪ྛ✀⫢⮚ᶵ⬟ࢆࣄࢺ⫢⣽⬊ྠᵝᫎࡋࡓࣉࣟࣇࣝࢆ♧ࡍ 㸦Gripon et al., 2002; Parent et al., 2004㸧㸬ࡲࡓ㸪HepaRG ⣽⬊ࡣศࡉࡏࡿࡇ࡛㸪⫢⸆≀௦ㅰ㓝 ⣲ࡀ㸪ࣄࢺ⫢⣽⬊ྠ➼ࣞ࣋ࣝⓎ⌧ࡋ࡚࠸ࡿ⣽⬊⣔࡛࠶ࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ㸦Gripon et al., 2002; Guillouzo et al., 2007; Aninat et al., 2006㸧㸬ࡇࢀࡽࡢࡇࡽ HepaRG ⣽⬊ࡣ⸆≀ືែ◊✲ࡸ ẘᛶ◊✲࡞࡛ࣄࢺ⫢⣽⬊ࡢ௦᭰ࡋ࡚⏝࡛ࡁࡿ⣽⬊⣔ࡋ࡚ὀ┠ࡉࢀ㸪⸆◊✲࡞⏝ ࡉࢀࡘࡘ࠶ࡿ㸬ᐇ㝿ࡇࢀࡲ࡛⸆≀௦ㅰヨ㦂࡛ࡢ᭷⏝ᛶ㛵ࡍࡿሗ࿌㸦Kanebratt and Andersson ,
2008(a); Lübberstedt et al., 2011㸧㸪⸆≀┦స⏝㛵ࢃࡿ CYP ㄏᑟ㸪CYP 㜼ᐖࡢホ౯㛵ࡍࡿሗ࿌
㸦Kanebratt and Andersson, 2008(b); Turpeinen et al., 2009; Anthérieu et al., 2010; Ogasawara et al.,
2016㸧㸪⸆≀ࡢࢡࣜࣛࣥࢫண ࡢ᭷⏝ᛶ㛵ࡍࡿሗ࿌࡞㸦Zanelli et al., 2012㸧ືែ◊✲㛵 ࢃࡿሗ࿌ࡀከࡃ࡞ࡉࢀ࡚ࡁ࡚࠸ࡿ㸬
HepaRG ⣽⬊ࢆ⏝࠸ࡓẘᛶホ౯㛵ࡍࡿ◊✲ࡶ࠸ࡃࡘ࡞ࡉࢀ࡚ࡁ࡚࠸ࡿ㸦Aninat et al., 2006; Jossé et al., 2008; Gerets et al., 2012㸧㸬⸆≀௦ㅰᶵ⬟ࢆࣄࢺ⫢⣽⬊ྠ➼ࣞ࣋ࣝಖ᭷ࡋ࡚࠸ࡿࡇ ࡽ㸪HepG2 ⣽⬊࡞ࡢࢭࣝࣛࣥẚ㍑ࡋ㸪ࡼࡾࣄࢺ⫢⣽⬊㏆࠸ẘᛶホ౯ࣔࢹࣝ⣽⬊⣔ࡋ࡚ ࡑࡢ᭷⏝ᛶࡀ᳨ドࡉࢀ࡚ࡁ࡚࠸ࡿࡀ㸪ࡑࡢHepaRG ⣽⬊ࡢ௦ㅰάᛶẘᛶⓎ⌧ࡢ㛵ಀࡸ HepaRG ⣽⬊ࢆ⏝࠸ࡓ௦ㅰⓗάᛶࢆࡋࡓẘᛶⓎ⌧ᶵᵓ㛵ࡍࡿ◊✲ࡣࡇࢀࡲ࡛࠶ࡲࡾࡉࢀ࡚ࡁ࡚࠸ ࡞࠸㸬ࡑࡇ࡛ᮏ◊✲࡛ࡣ㸪HepaRG ⣽⬊ࡀࡇࢀࡽ௦ㅰⓗάᛶࢆࡋ࡚Ⓨ⌧ࡍࡿ⫢㞀ᐖᛶࢆ᳨ฟ࣭ ホ౯࡛ࡁࡿ⣔࡛࠶ࡿ࠸࠺ほⅬ࡛㸪HepaRG ⣽⬊ࡢ⣽⬊ẘᛶホ౯⣔ࡋ࡚ࡢ᭷⏝ᛶ㛵ࡍࡿ◊ ✲ࢆ⾜ࡗࡓ㸬
2.
➨
2 ❶ ⫢⣽⬊ホ౯⣔ࡢ⸆≀௦ㅰ㓝⣲άᛶホ౯
2.1
➨
1 ⠇ ⥴ゝ
HepaRG ⣽⬊ࡣศࡉࡏࡿࡇ࡛ࣄࢺ⫢⣽⬊୪ࡳ⸆≀௦ㅰ㓝⣲άᛶࢆ᭷ࡋ࡚࠸ࡿࡇࡀ▱ࡽ ࢀࡿ㸦Lübberstedt et al., 2011㸧㸬ࡲࡓᰴࡉࢀࡓ⣽⬊࡛࠶ࡿࡓࡵࣄࢺ⫢⣽⬊ẚ㍑ࡋ㸪ࣟࢵࢺ㛫ᕪ ࡀᑡ࡞࠸ࡇࡀᮇᚅࡉࢀࡿࡀ㸪HepaRG ⣽⬊ࡢ⸆≀௦ㅰ㓝⣲άᛶࡘ࠸࡚」ᩘࣟࢵࢺẚ㍑ࡋ࡚ά ᛶࢆホ౯ࡋ࡚࠸ࡿሗ࿌ࡣᑡ࡞࠸㸬ᮏ❶࡛ࡣHepaRG ࡢศ࣭ᇵ㣴ᚋࡢ௦⾲ⓗ࡞⸆≀௦ㅰ㓝⣲άᛶ ࡢホ౯ࢆ⾜࠸㸪ࣄࢺ⤖⫢⣽⬊ཬࡧ௦⾲ⓗ࡞⫢⣽⬊ࡢᰴ⣽⬊࡛࠶ࡿHepG2 ⣽⬊ࡢ௦ㅰάᛶẚ ㍑ࡋࡓ㸬2.2
➨
2 ⠇ HepaRG ⣽⬊ࡢ⸆≀௦ㅰ㓝⣲άᛶホ౯
2.2.1 ➨1 㡯 HepaRG ⣽⬊ࡢศᇵ㣴᮲௳HepaRG ⣽⬊ࡣ✀ᚋ㸪ᑓ⏝ࡢᇵᆅ㸦HepaRG®MAINTENANCE/METABOLISM MEDIUM 620㸧
࡛ᇵ㣴ᚋ 4 ᪥㹼7 ᪥࡛⸆≀௦ㅰ⬟ࡀୖ᪼ࡋ㸪14 ᪥ᚋࡲ࡛άᛶࡀ⥔ᣢࡉࢀ㸪ヨ㦂⏝࡛ࡁࡿࡀ
㸦Manufacture protocol ཧ↷㸧㸪ᮏ◊✲࡛ࡢ HepaRG ⣽⬊ࡢᇵ㣴᮲௳ࢆ☜ㄆࡍࡿࡓࡵ㸪✀ᚋࡢᙧែ ほᐹཬࡧ⸆≀௦ㅰ㓝⣲άᛶࡢ ᐃࢆ⾜ࡗࡓ㸦Fig. 2-1㸧㸬
Figure 2-1 Differentiation of HepaRG cells and the determination of drug-metabolizing enzymes activities in the differentiated HepaRG cells.
ࡑࡢ⤖ᯝ㸪HepaRG ࡣ Day7 ࡛⫢⣽⬊ᵝࢆ♧ࡍ⣽⬊ཬࡧ⫹⟶ୖ⓶ᵝࢆ♧ࡍ⣽⬊ࡢඹᇵ㣴⣔ࡢࡼ࠺࡞
ᙧែࡀほᐹࡉࢀ㸦Fig. 2-2㸧㸪᪤ሗࡢ㏻ࡾ࡛࠶ࡗࡓ㸦Gripon et al., 2002; Andersson et al., 2012㸧㸬ࡲࡓ ⤒ⓗ㸪ࡼࡾ⫢⣽⬊ᵝᙧែࢆ♧ࡍ⣽⬊ࡀ᥋㏆㸪จ㞟ࡋ࡚࠸ࡿᵝᏊࡀほᐹࡉࢀࡓ㸬ྛ✀⸆≀௦ㅰ 㓝⣲άᛶ ᐃࡢ⤖ᯝࡽ㸪CYP1A2㸪CYP2B6㸪CYP2C19㸪CYP2D6㸪CYP3A4 άᛶࡘ࠸࡚ࡣ⤒ ⓗୖ᪼ࡍࡿࡇࡀ☜ㄆࡉࢀࡓ㸬CYP2C9㸪UGT㸪SULT ࡘ࠸࡚ࡣ Day7 ࡛᭱ࡶ㧗ࡃ㸪Day16 ࡲ࡛ᚎࠎపୗഴྥࡀㄆࡵࡽࢀࡓ㸦Fig. 2-3㸪Table 2-1㸧㸬ࡇࡢࡼ࠺⤒ⓗάᛶ್ࡢኚືࡀㄆ ࡵࡽࢀࡓࡀ㸪ࣄࢺ⤖⫢⣽⬊㸦3 ࣟࢵࢺ㸧ࡢ⸆≀௦ㅰ㓝⣲άᛶ㸦➨ 2 ❶㸪➨ 3 ⠇㸧ẚ㍑ࡋࡓ
Pre-culture
Day0 Day1 Day7 Day9 Day14 Day16
Observation &
Drug-metabolizing enzymes activities
Seed Differentiation medium containing 2% DMSO (Medium 620) Medium change 7.2 ¼ 104 cells/well/96-well plate HepaRG cells
Figure 2-2 Observation of morphology of differentiated HepaRG cells.
Table 2-1 Drug-metabolizing enzymes activities in the differentiated HepaRG cells
CYP1A2: Phenacetin-O-deethylase, CYP2B6: Bupropion hydroxylase, CYP2C9: Diclofenac 4-hydroxylase, CYP2C19: Mephenytoin 4'-hydroxylase, CYP2D6: Bufuralol 1-hydroxylase, CYP3A4: Midazolam 1'-hydroxylase, UGT: 7-hydroxycoumarin glucronidation,
SULT: 7-hydroxycoumarin sulfation.
*: The day thawing and seeding was count as Day 0
Day 7 7.65 ± 0.41 10.6 ± 2.3 3.79 ± 0.16 2.73 ± 0.39 2.33 ± 0.33 131 ± 15 1644 ± 24 20.5 ± 0.8 Day 9 8.99 ± 0.70 11.8 ± 1.4 3.07 ± 0.10 3.02 ± 0.18 3.71 ± 0.06 222 ± 19 1827 ± 17 22.2 ± 1.3 Day 14 11.5 ± 0.8 18.1 ± 1.5 2.52 ± 0.38 4.96 ± 0.43 3.71 ± 0.34 216 ± 4 1477 ± 44 18.3 ± 0.3 Day 16 10.7 ± 0.6 21.5 ± 1.6 2.59 ± 0.29 5.19 ± 0.20 3.78 ± 0.05 234 ± 25 1287 ± 71 17.6 ± 0.8 UGT SULT HepaRG Cell Time point*
Drug-metabolizing enzyme activity (pmol·min-1·mg protein-1)
Mean䚷±䚷SD
CYP1A2 CYP2B6 CYP2C9 CYP2C19 CYP2D6 CYP3A4
Day 7
x10
Day 9
Day 14
x20
Figure 2-3 Determination of drug-metabolizing enzymes activities at the several time points of 0 5 10 15 20 Human hepatocyte (3 Lot)
Day7 Day9 Day14 Day16
(p mo l· mi n -1·m g p rote in -1) CYP1A2 activity HepaRG 0 5 10 15 20 25 Human hepatocyte (3 Lot)
Day7 Day9 Day14 Day16
(p mo l· mi n -1·m g p rote in -1) CYP2B6 activity HepaRG 0 1 2 3 4 5 Human hepatocyte (3 Lot)
Day7 Day9 Day14 Day16
(p mo l· mi n -1·m g p rote in -1) CYP2C9 activity HepaRG 0 1 2 3 4 5 6 Human hepatocyte (3 Lot)
Day7 Day9 Day14 Day16
(p mo l· mi n -1·m g p rote in -1) CYP2C19 activity HepaRG 0 1 2 3 4 5 Human hepatocyte (3 Lot)
Day7 Day9 Day14 Day16
(p mo l· mi n -1·m g p rote in -1) CYP2D6 activity HepaRG 0 50 100 150 200 250 Human hepatocyte (3 Lot)
Day7 Day9 Day14 Day16
(p mo l· mi n -1·m g p rote in -1) CYP3A4 activity HepaRG 0 500 1000 1500 2000 Human hepatocyte (3 Lot)
Day7 Day9 Day14 Day16
(p mo l· mi n -1·m g p rote in -1) UGT activity HepaRG 0 5 10 15 20 25 Human hepatocyte (3 Lot)
Day7 Day9 Day14 Day16
(p mo l· mi n -1·m g p rote in -1) SULT activity HepaRG
2.2.2 ➨2 㡯 ศࡋࡓ HepaRG ⣽⬊ࡢ⸆≀௦ㅰ㓝⣲άᛶホ౯
2.2.1 ࡛ศ᮲௳ࡋ࡚タᐃࡋࡓ✀ᚋ๓ᇵ㣴 Day7 ࡢ HepaRG ⣽⬊ 3 ࣟࢵࢺࡘ࠸࡚㸪⸆≀௦ ㅰ㓝⣲άᛶࢆ ᐃࡋࡓ㸬ࡑࡢ⤖ᯝ㸪☜ㄆࡋࡓࡍ࡚ࡢศᏊ✀ࡘ࠸࡚άᛶࡀㄆࡵࡽࢀ㸪Ḟᦆࡋ࡚
࠸ࡿࡶࡢࡣㄆࡵࡽࢀ࡞ࡗࡓ㸬άᛶ್ࡢࣟࢵࢺ㛫ᕪࡶ࡛᭱ࡶCYP2B6㸪CYP2C19 ࠾ࡅࡿ⣙ 5
ಸ⛬ᗘ࡛࠶ࡾ㸪ࡁ࡞ࣟࢵࢺ㛫ᕪࡣㄆࡵࡽࢀ࡞ࡗࡓ㸦Table 2-2㸧㸬
Table 2-2 Drug-metabolizing enzymes activities in the differentiated HepaRG cells
CYP1A2: Phenacetin-O-deethylase, CYP2B6: Bupropion hydroxylase, CYP2C9: Diclofenac 4-hydroxylase, CYP2C19: Mephenytoin 4'-hydroxylase, CYP2D6: Bufuralol 1-hydroxylase, CYP3A4: Midazolam 1'-hydroxylase, UGT: 7-hydroxycoumarin glucronidation,
SULT: 7-hydroxycoumarin sulfation.
2.3
➨
3 ⠇ ࣄࢺ⫢ࡀࢇ⏤᮶⣽⬊ HepG2 ⣽⬊ࡢ⸆≀௦ㅰ㓝⣲άᛶホ౯
ศᚋࡢHepaRG ⣽⬊ࡢ⸆≀௦ㅰ㓝⣲άᛶẚ㍑ࡍࡿࡓࡵ㸪ࣄࢺ⫢ࡀࢇ⏤᮶⣽⬊ HepG2 ⣽⬊ࡢ
⸆≀௦ㅰ㓝⣲άᛶࢆ ᐃࡋࡓ㸬ࡑࡢ⤖ᯝ㸪☜ㄆࡋࡓࢇࡢ⸆≀௦ㅰ㓝⣲άᛶࡘ࠸᳨࡚ฟୗ
㝈௨ୗ࡛࠶ࡗࡓ㸬ࢇάᛶ್ࡢㄆࡵࡽࢀ࡞ࡗࡓPhase I 㓝⣲ẚ㍑ࡋ㸪Phase II 㓝⣲࡛࠶ࡿ㸪
UGT㸪SULT 㛵ࡋ࡚ࡣάᛶ್ࡀㄆࡵࡽࢀ㸪ࡇࢀࡽࡢࣉࣟࣇࣝࡣ㐣ཤࡢሗ࿌㸦Wilkeninget al.,
2003㸧ࡼࡿⓎ⌧㔞ྠᵝࡢࣉࣟࣇ࡛ࣝ࠶ࡗࡓ㸦Table 2-3㸧㸬
Table 2-3 Drug-metabolizing enzymes activities in the HepG2 cells
CYP1A2: Phenacetin-O-deethylase, CYP2B6: Bupropion hydroxylase, CYP2C9: Diclofenac 4-hydroxylase, CYP2C19: Mephenytoin 4'-hydroxylase, CYP2D6: Bufuralol 1-hydroxylase, CYP3A4: Midazolam 1'-hydroxylase, UGT: 7-hydroxycoumarin glucronidation, SULT: 7-hydroxycoumarin sulfation.
ND: Not detected
HepG2 ND ND ND ND 0.0150 ± 0.0101 0.225 ± 0.047 33.7 ± 5.1 20.8 ± 4.8
Cell
Drug metabolizing enzyme activity (pmol·min-1·mg protein-1)
Mean䚷±䚷SD
CYP1A2 CYP2B6 CYP2C9 CYP2C19 CYP2D6 CYP3A4 UGT SULT
HPR116062 7.65 ± 0.41 10.6 ± 2.3 3.79 ± 0.16 2.73 ± 0.39 2.33 ± 0.33 131 ± 15 1644 ± 24 20.5 ± 0.8
HPR116150 8.33 ± 1.23 34.4 ± 2.8 19.4 ± 4.9 6.21 ± 0.31 3.34 ± 0.48 114 ± 20 1962 ± 121 61.4 ± 2.3
HPR116169 10.2 ± 1.9 50.7 ± 3.9 12.5 ± 2.8 10.3 ± 0.9 2.21 ± 0.39 69.6 ± 11.9 2254 ± 253 61.8 ± 7.5
HepaRG
UGT SULT
Cell Lot No.
Drug metabolizing enzyme activity (pmol·min-1·mg protein-1)
Mean䚷±䚷SD
2.4
➨
4 ⠇ ࣄࢺ⤖⫢⣽⬊ࡢ⸆≀௦ㅰ㓝⣲άᛶホ౯
ศᚋࡢHepaRG ⣽⬊ࡢ⸆≀௦ㅰ㓝⣲άᛶẚ㍑ࡍࡿࡓࡵ㸪ࣄࢺ⫢⣽⬊ࡢ⸆≀௦ㅰ㓝⣲άᛶࢆ
ᐃࡋࡓ㸬ࣄࢺ⫢⣽⬊ࡋ࡚㸪ಶయูࡢࣄࢺ⤖⫢⣽⬊㸦Cryopreserved Human Hepatocyte㸪Plateable, BioreclamationIVT ♫㸧ࢆ 3 ࣟࢵࢺ㸦3 ࢻࢼ࣮㸧⏝ࡋࡓ㸬⤖⫢⣽⬊ࢆ✀㸪୍ᬌ᥋╔ᚋ㸪⸆≀ ௦ㅰ㓝⣲άᛶࢆ ᐃࡋࡓ㸬⤖⫢⣽⬊ࡣ㸪⼥ゎ࣭✀┤ᚋࡣ᭱ࡶ⸆≀௦ㅰ㓝⣲άᛶࡀ㧗ࡃ㸪✀ ᚋ㛫⤒㐣క࠸㸪άᛶ್ࡀపୗࡍࡿࡇࡀ୍⯡ⓗ▱ࡽࢀ࡚࠸ࡿࡀ㸪ᮏ ᐃ࡛ࡣ㸪⤖⫢⣽⬊ ࢆ⏝࠸ࡓ⣽⬊ẘᛶホ౯ࡢࢵࢭࢹࢨࣥྜࢃࡏ㸪✀୍࣭ᬌ᥋╔ᚋ㸪⸆≀᭚㟢ࢆ㛤ጞࡍࡿࢱ ࣑ࣥࢢ࡛άᛶࢆホ౯ࡋࡓ㸬ࡑࡢ⤖ᯝ㸪✀୍ᬌ᥋╔ᚋࡶ⸆≀௦ㅰ㓝⣲άᛶࡣㄆࡵࡽࢀࡓࡀ㸪ࣟ
ࢵࢺSHM 㛵ࡋ࡚ࡣ CYP2B6, CYP2C19 ࡘ࠸࡚ࡣ᳨ฟୗ㝈௨ୗ࡛࠶ࡗࡓ㸬ࡲࡓ CYP3A4 ࡘ
࠸࡚ࡶ㸪᭱ࡶάᛶࡢ㧗ࡗࡓࣟࢵࢺYEM ẚ㍑ࡋ㸪⣙ 200 ಸ⛬ᗘάᛶ್ࡀప࠸࠸࠺⤖ᯝ࡛࠶
ࡗࡓ㸬ࡇࡢ⤖ᯝࡣῧࡢࢹ࣮ࢱࢩ࣮ࢺ㸦Certificate of analysis㸧ྠᵝࡢഴྥ࡛࠶ࡾ㸪ࢻࢼ࣮ࡢಶ య⏤᮶ࡍࡿάᛶ್ࡢᕪ࡛࠶ࡿࡇࡀ♧၀ࡉࢀࡓ㸦Table 2-4㸧㸬
Table 2-4 Drug-metabolizing enzymes activities in the cryopreserved human hepatocytes
CYP1A2: Phenacetin-O-deethylase, CYP2B6: Bupropion hydroxylase, CYP2C9: Diclofenac 4-hydroxylase, CYP2C19: Mephenytoin 4'-hydroxylase, CYP2D6: Bufuralol 1-hydroxylase, CYP3A4: Midazolam 1'-hydroxylase,UGT: 7-hydroxycoumarin glucronidation,
SULT: 7-hydroxycoumarin sulfation, ND: Not detectable SHM 2.06 ± 1.31 1.81 ± 0.40 0.178 ± 0.038 0.349 ± 0.132 255 ± 94 22.7 ± 6.4 CPQ 1.94 ± 0.44 1.66 ± 0.34 1.84 ± 0.33 2.49 ± 0.59 0.908 ± 0.149 33.7 ± 5.7 391 ± 76 7.12 ± 1.02 YEM 4.34 ± 1.05 2.61 ± 0.68 3.72 ± 0.65 0.788 ± 0.185 0.660 ± 0.175 72.6 ± 16.7 342 ± 62 6.24 ± 1.31 CHHs ND ND UGT SULT
Cell Lot No.
Drug metabolizing enzyme activity (pmol·min-1·mg protein-1)
Mean䚷±䚷SD
2.5
➨
5 ⠇ ᑠᣓ
ศࡋࡓHepaRG ⣽⬊ࡢ⸆≀௦ㅰ㓝⣲άᛶࡢࣉࣟࣇࣝࢆ☜ㄆࡍࡿࡓࡵ㸪HepaRG㸪HepG2㸪 ࣄࢺ⤖⫢⣽⬊ࡢάᛶ್ࢆ ᐃࡋ㸪ẚ㍑ࡋࡓ㸦Fig. 2-4㸧㸬ࡑࡢ⤖ᯝ㸪HepaRG ⣽⬊௦ㅰ㓝⣲άᛶࡣ ࣄࢺ⤖⫢⣽⬊ࡢ㓝⣲άᛶẚ㍑ࡋ㸪☜ㄆࡋࡓࡍ࡚ࡢศᏊ✀ࡘ࠸࡚㸪ྠ➼௨ୖࡢάᛶ࡛࠶ࡗ ࡓ㸬ࡇࡢࡇࡣࣄࢺึ௦⫢⣽⬊άᛶࢆẚ㍑ࡋࡓ㐣ཤࡢሗ࿌ࡶ୍⮴ࡋ࡚࠸ࡿ㸦Lübberstedt et al., 2011㸧㸬ࡲࡓࣄࢺ⤖⫢⣽⬊ࡣศᏊ✀ࡼࡗ࡚ࡣάᛶࡀⴭࡋࡃప࠸㸪ࡲࡓࡣ᳨ฟୗ㝈௨ୗࡢࣟࢵ ࢺࡀㄆࡵࡽࢀࡓࡀ㸪HepaRG ⣽⬊ࡣ☜ㄆࡋࡓศᏊ✀ࡢࡍ࡚ࡘ࠸࡚άᛶ್ࢆಖᣢࡋ࡚࠾ࡾ㸪 ᅇ☜ㄆࡋࡓ3 ࣟࢵࢺࡘ࠸࡚ࡔࡅ࡛ࡶ㸪ࣟࢵࢺ㛫࡛άᛶ್ࡘ࠸࡚᭱ 200 ಸ㸦CYP3A4㸧⛬ᗘࡢᕪࡀㄆࡵࡽࢀࡓࡢᑐࡋ࡚㸪HepaRG ⣽⬊ࡣࣟࢵࢺ㛫ᕪࡀ࡛᭱ࡶ 5 ಸ⛬ᗘ㸦CYP2B6, CYP2C19㸧
࡛࠶ࡗࡓ㸬⸆≀௦ㅰ㓝⣲άᛶࡢࣟࢵࢺ㛫ᕪࡀẚ㍑ⓗᑡ࡞ࡃ㸪⫢⣽⬊ホ౯⣔ࡋ࡚Ᏻᐃࡋࡓ௦ㅰ㓝 ⣲άᛶࢆಖ᭷ࡋ࡚࠸ࡓ㸬
HepaRG ⣽⬊ࡢ CYP2D6 ࡢάᛶࡘ࠸࡚ࡣ㸪ሗ࿌࠶ࡿࡼ࠺㸪ࢻࢼ࣮ࡢಶయ⏤᮶ࡍࡿ poor metabolizer ࡛࠶ࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿࡀ㸦Gripon et al., 2002; Guillouzo et al., 2007㸧㸪ᅇࡢ HepaRG
⣽⬊ࡢάᛶ ᐃࡢ⤖ᯝ࡛ࡣCYP2D6 ࡢᇶ㉁࡛࠶ࡿ Bufuralol ࡢ௦ㅰ≀࡛࠶ࡿ 1-hydroxybufuralol ࡢ
⏕ᡂࡣㄆࡵࡽࢀ㸪άᛶ್ࡣࣄࢺ⤖⫢⣽⬊ྠ➼࡛ࣞ࣋ࣝ࠶ࡗࡓ㸬ᅇࡢ ᐃࡢ⤖ᯝࢆ⣽⬊ᩘ⿵
ṇࡋࡓ⤖ᯝࡣ0.514 – 1.03 pmol/min/106cells㸦ᇶ㉁⃰ᗘ㸸5Pmol/L bufuralol㸧࡛࠶ࡗࡓ㸬ྠࡌᇶ㉁
ࢆ⏝࠸ࡓሗ࿌࡛ࡣ⣙ 3.0 pmol/min/106cells㸦ᇶ㉁⃰ᗘ㸸25 Pmol/L bufuralol㸧࠸࠺⤖ᯝ࡛࠶ࡾ
㸦Lübberstedt et al., 2011㸧㸪ᇶ㉁⃰ᗘࡢ㐪࠸ࢆ⪃៖ࡍࡿ㸪ᅇࡢ HepaRG ⣽⬊ࡢ⤖ᯝࡣᙜヱሗ࿌
ྠ➼ࣞ࣋ࣝࡢάᛶ࡛࠶ࡿᛮࢃࢀࡿ㸬ࡲࡓྠሗ࿌࡛ࡣ☜ㄆࡋࡓࣄࢺึ௦⫢⣽⬊HepaRG ⣽⬊
ࡢBufuralol Ỉ㓟άᛶࡣྠ➼࡛ࣞ࣋ࣝ࠶ࡗࡓሗ࿌ࡋ࡚࠸ࡿ㸬
HepG2 ⣽⬊ࡘ࠸࡚ ᐃࡋࡓ௦ㅰ㓝⣲άᛶࡢ⤖ᯝࡽࡣ㸪᪤ሗࡢ㏻ࡾ㸦Wilkening et al., 2003;
Westerink et al., 2007(a); Westerink et al., 2007(b)㸧㸪SULT ࡢάᛶࡘ࠸࡚ࡣ㸪ࣄࢺ⤖⫢⣽⬊ྠ➼
ࣞ࣋ࣝࡢάᛶࢆಖ᭷ࡋ࡚࠸ࡓࡀ㸪ࢇࡢศᏊ✀ࡢ⸆≀௦ㅰ㓝⣲ࡘ࠸࡚ࡣࣄࢺ⤖⫢⣽⬊ ẚ㍑ࡋ㸪άᛶࡀⴭࡋࡃప࠸ࡲࡓࡣ㸪᳨ฟୗ㝈௨ୗ࡛࠶ࡗࡓ㸬HepG2 ẚ࡚ HepaRG ⣽⬊ࡢ⸆≀ ௦ㅰ㓝⣲άᛶࡣ༑ศ㧗ࡃ㸪HepaRG ⣽⬊ࡣ⫢⮚ࡢ≉ᚩⓗ࡞ᶵ⬟࡛࠶ࡿ⸆≀௦ㅰ㓝⣲άᛶࢆ༑ศ
Figure 2-4 Comparison of drug metabolizing enzyme activities in HepG2 cells, cryopreserved human hepatocytes, and HepaRG cells.
Three different lots of CHHs (Lot. SHM, CPQ, and YEM) and HepaRG cells (Lot. HPR116062, HPR116150, and HPR116169) were examined. Results of HepG2 cell analysis: gray bars are means r standard deviation (S.D., n 8). Results of CHHs: black bars are means r S.D.; Lot. SHM: n 6, CPQ: n 3,
0.0 3.0 6.0 9.0 12.0 15.0 HepG2 SHM CPQ YEM 062 150 169 P h en acetin-O-d eet h yla se (p mo l· mi n -1·m g pr otei n -1) CYP1A2 activity
Human hepatocytes HepaRG
ND (a) 0.0 10.0 20.0 30.0 40.0 50.0 60.0 HepG2 SHM CPQ YEM 062 150 169 B u pr opi on h ydr ox yl ase (pm o l· m in -1·m g pr otei n -1) CYP2B6 activity
Human hepatocytes HepaRG ND ND (b) 0.0 10.0 20.0 30.0 HepG2 SHM CPQ YEM 062 150 169 D iclofen ac 4 މ-h ydr o xy lase (pm o l· m in -1·m g pr otei n -1) CYP2C9 activity
Human hepatocytes HepaRG
ND (c) 0.0 3.0 6.0 9.0 12.0 15.0 HepG2 SHM CPQ YEM 062 150 169 Meph en ytoi n 4' -h ydr o xy la se (pm o l· m in -1·m g pr otei n -1) CYP2C19 activity
Human hepatocytes HepaRG
ND ND (d) 0 50 100 150 200 HepG2 SHM CPQ YEM 062 150 169 M id azo la m 1 މ-h ydr o xy lase (pm o l· m in -1·m g pr otei n -1) CYP3A4 activity
Human hepatocytes HepaRG
0.225 0.349 (f) 0 500 1000 1500 2000 2500 3000 HepG2 SHM CPQ YEM 062 150 169 7-hy dr ox y coum ar in gluc ronida tion (p m ol· m in -1·m g pr ot ei n -1) UGT activity
Human hepatocytes HepaRG 33.7 (g) 0 20 40 60 80 100 HepG2 SHM CPQ YEM 062 150 169 7-hy dr ox y coum ar in s ulf at ion (p m ol· m in -1·m g pr ot ei n -1) SULT activity
Human hepatocytes HepaRG
(h) 0.0 1.0 2.0 3.0 4.0 5.0 HepG2 SHM CPQ YEM 062 150 169 B u fu ra lo l 1 މ-h ydr o xy lase (p mo l· mi n -1·m g pr otei n -1) CYP2D6 activity
Human hepatocytes HepaRG
0.0150
3.
➨
3 ❶ ⫢ẘᛶ≀㉁ࢆ⏝࠸ࡓ⣽⬊ẘᛶホ౯
3.1
➨
1 ⠇ ⥴ゝ
ศࡋࡓHepaRG ⣽⬊ࡣࣄࢺ⫢⣽⬊ྠ➼ࣞ࣋ࣝࡢ⸆≀௦ㅰ⬟ࢆಖ᭷ࡋ࡚࠸ࡿࡇࡀ♧ࡉࢀࡓ ࡀ㸪ࡇࡢHepaRG ⣽⬊ࡀ⫢ẘᛶ≀㉁ࡢ⫢㞀ᐖᛶࢆホ౯ࡍࡿࣄࢺ⫢⣽⬊ホ౯⣔ࡋ࡚᭷⏝࡛࠶ࡿ ࢆ᳨ウࡍࡿࡓࡵ㸪5 ࡘࡢ௦⾲ⓗ࡞⫢ẘᛶ≀㉁ࢆ⏝࠸࡚⣽⬊ẘᛶࡢホ౯ࢆ⾜࠸㸪ࡑࡢ⣽⬊㞀ᐖᛶࢆ ࣄ ࢺ ⤖ ⫢ ⣽ ⬊ ẚ ㍑ ࡋ ࡓ 㸬 ⫢ ẘ ᛶ ≀ ㉁ ࡋ ࡚ ⏝ ࠸ ࡓ 5 ࡘ ࡢ ྜ ≀ 㸦 Aflatoxin B1 㸪 Cyclophosphamide㸪Acetaminophen㸪Troglitazone㸪Tamoxifen㸧ࡣ㸪CYP ࡼࡿ௦ㅰⓗάᛶࡼ ࡗ࡚ẘᛶⓎ⌧ࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ㸬3.2
➨
2 ⠇ ࣄࢺ⤖⫢⣽⬊ࢆ⏝࠸ࡓ⣽⬊ẘᛶホ౯
ศࡋࡓHepaRG ⣽⬊ࡣࣄࢺ⫢⣽⬊ྠ➼ࣞ࣋ࣝࡢ⸆≀௦ㅰ⬟ࢆಖ᭷ࡋ࡚࠸ࡿࡇࡀ♧ࡉࢀࡓ ࡀ㸪ࡇࡢHepaRG ⣽⬊ࡀ⫢ẘᛶ≀㉁ࡢ⫢㞀ᐖᛶࢆホ౯ࡍࡿࣄࢺ⫢⣽⬊ホ౯⣔ࡋ࡚᭷⏝࡛࠶ࡿ ࢆ᳨ウࡍࡿࡓࡵ㸪ࣄࢺ⤖⫢⣽⬊ࢆ⏝࠸࡚⫢ẘᛶ≀㉁ࡼࡿ⣽⬊ẘᛶホ౯ࢆ⾜ࡗࡓ㸬ࣄࢺ⤖⫢ ⣽⬊ࡣ➨ 2 ❶㸪➨ 4 ⠇࡛⸆≀௦ㅰ㓝⣲άᛶࢆホ౯ࡋࡓࣄࢺ⤖⫢⣽⬊㸦Cryopreserved Human Hepatocyte㸪Plateable, BioreclamationIVT ♫㸧ࢆ 3 ࣟࢵࢺ㸦3 ࢻࢼ࣮㸧⏝ࡋࡓ㸬ྜ≀ࢆ᭚㟢ࡋ48 㛫ᚋ⣽⬊㞀ᐖᛶࢆホ౯ࡋࡓ㸬⣽⬊㞀ᐖᛶࡢホ౯ࡣ ATP ࢵࢭ㸦CellTiter-GloTM㸪Promega㸧
ࢆ⏝࠸ࡓ㸬
ࡑࡢ⤖ᯝࢆTable 3-1 ♧ࡍ㸬AFB1㸪CPA㸪TAM ࡘ࠸࡚ࡣࡢࣟࢵࢺࡢ⤖⫢⣽⬊ᑐࡋ࡚
ࡶ⃰ᗘ౫Ꮡⓗ࡞⣽⬊㞀ᐖᛶࡀㄆࡵࡽࢀࡓ㸬APAP ࡘ࠸࡚ࡣ㸪ࣟࢵࢺ CPQ ࡘ࠸࡚ࡢࡳ㸪᭱㧗⃰
ᗘࡢ10000Pmol/L ࠾࠸࡚⣽⬊㞀ᐖᛶࡀࢃࡎ☜ㄆࡉࢀࡓࡀ㸦Cell viability: 91.6%㸧㸪2 ࣟࢵࢺ
ࡘ࠸࡚ࡣᅇホ౯ࡋࡓ᭱㧗⃰ᗘࡲ࡛⣽⬊㞀ᐖᛶࡣㄆࡵࡽࢀ࡞ࡗࡓ㸬TGZ ࡘ࠸࡚ࡣ㸪3 ࣟࢵ
ࢺࡶᅇホ౯ࡋࡓ᭱㧗⃰ᗘ100Pmol/L ࡲ࡛⣽⬊㞀ᐖᛶࡣㄆࡵࡽࢀ࡞ࡗࡓ㸬
Table 3-1 Results of cytotoxicity assays of five hepatotoxicants in cryopreserved human hepatocytes
AFB1: Aflatoxin B1, CPA: Cyclophosphamide, APAP: Acetaminophen, TAM: Tamoxifen, TGZ: Troglitazone, Conc.: Concentration
IC50 IC50 IC50
(Pmol/L) Mean S.D. (Pmol/L) Mean S.D. (Pmol/L) Mean S.D. (Pmol/L)
0 100.0 2.6 100.0 2.4 100.0 2.4 0.2 117.5 2.8 - - - -0.6 122.7 3.4 112.7 1.5 107.9 2.3 2 120.0 3.7 118.7 1.1 89.1 2.6 6 85.3 2.6 82.7 2.5 38.1 1.5 20 18.4 1.1 59.4 3.3 6.2 0.7 0 100.0 2.0 100.0 1.0 100.0 1.3 250 100.1 2.1 - - - -750 101.3 1.9 96.9 0.4 101.3 1.8 2,500 105.3 0.8 98.0 1.8 99.4 1.0 7,500 107.7 1.9 94.9 1.4 93.5 2.7 25,000 79.8 1.1 91.2 1.8 50.4 2.0 0 100.0 3.4 100.0 2.4 100.0 2.2 100 100.6 3.2 - - - -300 102.1 2.2 94.6 3.0 101.8 2.9 1,000 102.0 1.3 94.1 4.0 99.2 2.6 3,000 103.5 1.2 90.1 4.9 94.9 3.5 10,000 98.6 0.8 91.6 0.2 104.9 9.8 0 100.0 3.5 100.0 1.6 100.0 4.3 1 100.5 1.0 - - - -3 100.4 1.4 103.2 3.3 107.0 2.1 10 98.8 2.8 103.8 4.2 107.8 1.0 30 95.0 1.6 102.9 2.1 102.8 3.9 100 96.4 0.8 99.1 1.3 98.0 2.6 0 100.0 3.1 100.0 0.8 100.0 0.3 1 100.0 1.6 - - - -3 100.4 1.0 94.3 1.5 100.6 2.1 10 96.5 2.9 86.3 1.9 97.0 2.5 30 67.5 3.7 73.8 3.9 81.8 2.3 100 0.2 0.0 0.3 0.0 0.2 0.0 AFB1 CPA APAP TGZ TAM Cell viability (% of control) Lot. SHM Compound Conc. Lot. CPQ Cell viability (% of control) 7.8 Cell viability (% of control) >25,000 >10,000 >100 40.3 䍦20 >25,000 >10,000 >100 54.4 4.7 䍦25,000 >10,000 >100 60.9 Lot. YEM
3.3
➨
3 ⠇ HepaRG ⣽⬊ࢆ⏝࠸ࡓ⣽⬊ẘᛶホ౯
➨3 ❶㸪➨ 2 ⠇࡛ࣄࢺ⫢⣽⬊ࡢ 5 ✀ࡢ⫢ẘᛶ≀㉁ࡢ⣽⬊ẘᛶࢆホ౯ࡋࡓࡀ㸪ྠᵝࡢ⫢ẘᛶ≀
㉁ࡢ HepaRG ⣽⬊ᑐࡍࡿ⣽⬊ẘᛶࢆホ౯ࡋࡓ㸬ศࡋࡓ HepaRG ⣽⬊⫢ẘᛶ≀㉁㸦AFB1㸪
CPA㸪APAP㸪TGZ㸪TAM㸧ࢆ᭚㟢ࡋ㸪48 㛫ᚋ⣽⬊㞀ᐖᛶࢆホ౯ࡋࡓ㸬⣽⬊㞀ᐖᛶࡢᣦᶆ
ࡣATP ࢵࢭࢆ⏝࠸ࡓ㸬⏝ࡋࡓࣟࢵࢺࡣ⸆≀௦ㅰάᛶࡀࡢࣟࢵࢺࡶྠ➼࡛ࣞ࣋ࣝ࠶ࡗࡓࡢ
࡛㸪௵ពࡢ1 ࣟࢵࢺ㸦HPR116150㸧ࢆ⏝ࡋࡓ㸬
ࡑࡢ⤖ᯝࢆTable 3-2 ♧ࡍ㸬ࡍ࡚ࡢྜ≀ࡘ࠸࡚⃰ᗘ౫Ꮡⓗ⣽⬊㞀ᐖᛶࡀ☜ㄆࡉࢀࡓ㸬
⣽⬊ẘᛶࡢIC50ࡀ⟬ฟࡉࢀࡓྜ≀ࡀAFB1㸦IC50=5.0Pmol/L㸧㸪TGZ㸦IC50=80.0 Pmol/L㸧㸪TAM
㸦IC50=15.8Pmol/L㸧࡛࠶ࡾ㸪CPA APAP ࡘ࠸࡚ࡣ IC50ࡀ⟬ฟࡉࢀ࡞ࡗࡓࡀ㸪᭱㧗⃰ᗘࡢ⣽
⬊ẘᛶࡀࡑࢀࡒࢀCPA ࡘ࠸࡚ࡣ 25000Pmol/L ࡛ 56.7%㸪APAP ࡘ࠸࡚ 10000 Pmol/L ࡛ 75.4%
࡛࠶ࡾ⣽⬊㞀ᐖᛶࡀ☜ㄆࡉࢀࡓ㸬
Table 3-2 Results of cytotoxicity assays of five hepatotoxicants in HepaRG Cells
AFB1: Aflatoxin B1, CPA: Cyclophosphamide, APAP: Acetaminophen, TAM: Tamoxifen, TGZ: Troglitazone, Conc.: Concentration.
IC50
(Pmol/L) Mean S.D. (Pmol/L)
0 100.0 2.3 0.2 96.7 3.3 0.6 87.3 3.9 2 76.4 3.1 6 43.7 1.1 20 10.5 0.6 0 100.0 3.4 250 99.1 1.3 750 98.9 0.6 2,500 99.3 1.6 7,500 95.9 2.4 25,000 56.7 1.1 0 100.0 1.5 100 97.4 3.2 300 97.7 2.6 1,000 96.1 3.6 3,000 95.8 4.6 10,000 75.4 2.0 0 100.0 1.6 1 98.8 1.6 3 98.7 1.3 10 98.1 2.1 30 96.4 1.0 100 0.3 0.0 0 100.0 3.1 1 100.5 0.4 3 98.0 0.5 10 98.0 1.1 30 0.3 0.0 100 0.1 0.0 AFB1 CPA Compound APAP TGZ TAM Conc. 5.0 䍦25,000 >10,000 80.0 15.8 Lot. HPR116150 Cell viability (% of control)
3.4
➨
4 ⠇ ᑠᣓ
HepaRG ⣽⬊ࡢ⫢ẘᛶホ౯⣔ࡋ࡚ࡢ᭷⏝ᛶࢆ☜ㄆࡍࡿࡓࡵ㸪௦ㅰⓗάᛶࡼࡾẘᛶࢆⓎ⌧
ࡍࡿࡇࡀ▱ࡽࢀࡿ5 ✀ࡢ⫢ẘᛶ≀㉁ࡘ࠸࡚㸪HepaRG ⣽⬊ࢆ⏝࠸࡚⣽⬊ẘᛶࢆホ౯ࡋ㸪ࣄࢺ
⤖⫢⣽⬊ࡢẘᛶࡢឤཷᛶẚ㍑ࡋࡓ㸦Fig. 3㸧㸬ࡑࡢ⤖ᯝ㸪AFB1 ࡘ࠸࡚ࡣ HepaRG ⣽⬊ IC50=5.0
Pmol/L㸪ࣄࢺ⤖⫢⣽⬊㸦3 ࣟࢵࢺ㸧࡛ࡣ IC50=4.7 Pmol/L㹼⣙ 20 Pmol/L ࣄࢺ⤖⫢⣽⬊ࡢឤ
ཷᛶࡰྠ➼࡛࠶ࡗࡓ㸬ࣄࢺ⤖⫢⣽⬊ࡢ୰࡛᭱ࡶ AFB1 ࡢឤཷᛶࡀ㧗ࡗࡓࣟࢵࢺ YEM
ࡣAFB1 ࡢẘᛶ㛵ಀࡍࡿࡇ࡛▱ࡽࢀࡿ௦ㅰ≀ AFB1-8,9-epoxide యࡢ⏕ᡂ㛵ࡍࡿ௦ㅰ㓝⣲
CYP1A2, CYP3A4 ࡢάᛶࡀ᭱ࡶ㧗࠸ࣟࢵࢺ࡛࠶ࡾ㸪HepaRG ⣽⬊ࡶྠ➼ࡢ௦ㅰάᛶ࡛࠶ࡾ㸪⣽⬊
ẘᛶࡢឤཷᛶࡶ᭱ࡶ㏆ࡋࡗࡓ㸬CPA ࡶ᭱ࡶ௦ㅰάᛶࡀ㧗ࡗࡓࣟࢵࢺ YEM ࡛⣽⬊ẘᛶࡀ㧗ࡃ㸪
HepaRG ⣽⬊᭱ࡶ⣽⬊ẘᛶࡢឤཷᛶࡀ㏆ࡋࡗࡓ㸬᭱ࡶࣄࢺ⤖⫢⣽⬊ HepaRG ⣽⬊࡛ឤཷ
ᛶࡀ␗࡞ࡗࡓࡢࡣ TGZ ࡢឤཷᛶࡢ㐪࠸࡛࠶ࡾ㸪ࣄࢺ⤖⫢⣽⬊࡛ࡣ 3 ࣟࢵࢺࡶ᭱㧗⃰ᗘࡢ
100 Pmol/L ࡛⣽⬊㞀ᐖᛶࡀㄆࡵࡽࢀ࡞ࡗࡓࡢᑐࡋ࡚㸪HepaRG ⣽⬊࡛ࡣ 100 Pmol/L ࡛ Cell
viability 0.3%ࢇṚ⁛ࡋ࡚࠸ࡿ࠸࠺⤖ᯝ࡛࠶ࡗࡓ㸦IC50=80 Pmol/L㸧㸬ࡇࡢ⤖ᯝࡣ⌧ᛶ
ࢆ☜ㄆࡋ࡚ࡶྠᵝࡢ⤖ᯝ࡞ࡾ㸪ࡇࡢ⤖ᯝࡢ㐪࠸ࡘ࠸࡚ヲ⣽ࡢ᳨ウࡣᮍᐇ࡛ࡣ࠶ࡿࡀ㸪 HepaRG ⣽⬊ࡣ SULT ࡢ௦ㅰάᛶࡀ㧗ࡃ㸪TGZ ࡣẘᛶⓎ⌧㸪CYP ࡼࡿ௦ㅰάᛶࡢࡳ࡞ࡽࡎ㸪 ◲㓟ᢪྜయࡢ⣽⬊ẘᛶࡶ▱ࡽࢀࡿࡓࡵ㸦Funk et al., 2001; Saha et al., 2010㸧㸪ࡑࡢᙳ㡪ࡢྍ⬟ᛶࡶ
♧၀ࡉࢀࡓ㸬ࡇࢀࡽࡢࡇࡽ㸪☜ㄆࡋࡓ5 ࡘࡢ⫢ẘᛶ≀㉁ࡘ࠸࡚ HepaRG ⣽⬊ࡣࣄࢺ⤖⫢
Figure 3 Cytotoxicity of hepatotoxicants in cryopreserved human hepatocytes and HepaRG cells.
Comparative cytotoxic effects of AFB1, CPA, APAP, TGZ, and TAM on cryopreserved human hepatocytes (CHHs) and HepaRG cells. Results of CHHs are open symbols (Lot. SHM, Lot. CPQ, Lot. YEM). Results of CHHs are closed symbols (Lot. HPR116150). CHHs and HepaRG cells were exposed to test compounds for 48 h. Cell viability was evaluated using CellTiter-GloTM luminescent cell viability
assay. Results are normalized to control cells levels and expressed as means r standard deviation (S.D., n 3 cultures). The Figure 3 shows the summarized results of Table 3-1 and Table 3-2.
0 50 100 150 0.1 1 10 100 Ce ll v ia b ilit y ( % of c o nt rol ) Concentration (PPmol/L) Hepatocyte: SHM Hepatocyte: CPQ Hepatocyte: YEM HepaRG (a) 0 50 100 150 100 1000 10000 100000 Ce ll v ia b ilit y ( % of c ont rol ) Concentration (PPmol/L) Hepatocyte: SHM Hepatocyte: CPQ Hepatocyte: YEM HepaRG (b) 0 50 100 150 100 1000 10000 100000 Ce ll v ia b ilit y ( % of c o nt rol ) Concentration (PPmol/L) Hepatocyte: SHM Hepatocyte: CPQ Hepatocyte: YEM HepaRG (c) 0 50 100 150 1 10 100 Ce ll v ia b ilit y ( % of c o nt rol ) Concentration (PPmol/L) Hepatocyte: SHM Hepatocyte: CPQ Hepatocyte: YEM HepaRG (d) 0 50 100 150 1 10 100 Ce ll v ia b ilit y ( % of c o nt rol ) Concentration (PPmol/L) Hepatocyte: SHM Hepatocyte: CPQ Hepatocyte: YEM HepaRG (e)
Aflatoxin B1 Cyclophosphamide Acetaminophen
4.
➨
4 ❶ ⫢ẘᛶ≀㉁ࢆ⏝࠸ࡓ⥙⨶ⓗ㑇ఏᏊⓎ⌧ኚືゎᯒ
4.1
➨
1 ⠇ ⥴ゝ
⫢ẘᛶࡢẘᛶⓎ⌧ࡢᶵᗎゎ᫂ࡢᡭἲࡢ୍ࡘࡋ࡚⥙⨶ⓗ㑇ఏᏊⓎ⌧ኚືゎᯒ࡞ࡢᡭἲࡀ⏝࠸ ࡽࢀࡿ㸬ྜ≀ࢆ᭚㟢ࡍࡿࡇࡼࡾኚືࡍࡿ㑇ఏᏊࢆ⥙⨶ⓗゎᯒࡋ㸪up-regulate ཪࡣ down-regulate ࡋ࡚࠸ࡿ㑇ఏᏊࢆᢳฟࡋ㸪άᛶཪࡣᢚไࡉࢀ࡚࠸ࡿ㑇ఏᏊ⩌࣭ࣃࢫ࢙࢘ࢆゎᯒ ࡋ㸪ẘᛶⓎ⌧㛵ಀࡋ࡚࠸ࡿ࣓࢝ࢽࢬ࣒ࢆ᥎ᐃࡍࡿᡭἲ࡛࠶ࡿ㸬ࡇࡢࡼ࠺࡞ᡭἲ࡛⫢ẘᛶⓎ⌧ᶵ ᗎゎᯒࢆin vitro ࡢヨ㦂⣔࡛⾜࠺ሙྜ㸪᭷⏝࡞⫢⣽⬊ࣔࢹࣝヨ㦂⣔ࡀᚲせ࡞ࡿ㸬ࡇࢀࡲ࡛ HepG2 ࡞ࡢᰴࡉࢀࡓ⫢ࡀࢇ⣽⬊ࡣࣟࢵࢺ㛫ᕪ➼ࢆ⪃៖ࡍࡿᚲせࡀ࡞ࡃỗ⏝ᛶ㧗ࡃ⏝࠸ࡽࢀ࡚ࡁࡓ ࡀ㸪⸆≀௦ㅰ⬟ࡀపࡃ㸪⫢⮚ࡢ௦ㅰࢆᫎࡉࢀࡓ⣔ࡋ࡚༑ศ࡛࠶ࡿ㸬ࡑࡇ࡛ᮏ❶࡛ࡣHepaRG ⣽⬊ࡀ⥙⨶ⓗ㑇ఏᏊⓎ⌧ኚືゎᯒࡢᡭἲࢆ⏝࠸࡚㸪⫢ẘᛶ≀㉁ࡢ࣓࢝ࢽࢬ࣒ゎᯒ⏝ࡍࡿࡇ ࡀ࡛ࡁࡿホ౯⣔࡛࠶ࡿࡢ᳨ウࢆ⾜ࡗࡓ㸬⫢ẘᛶ≀㉁ࡋ࡚⣽⬊ẘᛶホ౯ࢆ⾜ࡗࡓྜ≀ࡢ࠺ࡕ㸪 CYP1A2, CYP3A4 ➼ࡼࡾ௦ㅰࡉࢀ⏕ᡂࡍࡿ epoxide యࡀẘᛶࢆ♧ࡍ AFB1㸪CYP2B6, CYP3A4➼ࡼࡾ⏕ᡂࡍࡿ4-hydroxy యࡀ㸪acrolen ࡞ࡢࡼࡾ⣽⬊㞀ᐖᛶࡢ࠶ࡿ௦ㅰ≀ࡀ⏕ᡂࡍࡿࡇࡀ
▱ࡽࢀ࡚࠸ࡿCPA ࢆホ౯ྜ≀ࡋ࡚⏝ࡋࡓ㸦Fig. 4-1㸧㸬
Figure 4-1 Metabolic pathway of aflatoxin B1 and cyclophosphamide and the toxic metabolites
Aflatoxin B1-exo -8,9-epoxide
Aflatoxin B1 CYP1A2 CYP3A4 CYP2A13 O O H H O CH3 O O O O O H H O CH3 O O O O NH P O O N Cl Cl NH P O O N Cl Cl OH NH2 P O O N Cl Cl O H CH2 O NH2 P O N Cl Cl O CYP2B6 CYP3A4 CYP2C9
Cyclophosphamide 4-Hydroxycyclophosphamide Aldophosphamide Acrolein
Phosphamide mustard Reactive metabolites (Cytotoxic) (a): Aflatoxin B1 (b): Cyclophosphamide Reactive metabolites (Cytotoxic) Reactive metabolites (Cytotoxic)
4.2
➨
2 ⠇ HepaRG ⣽⬊ཬࡧ HepG2 ⣽⬊ࢆ⏝࠸ࡓ㑇ఏᏊⓎ⌧ゎᯒ
ศࡉࡏࡓHepaRG ⣽⬊ཬࡧ㸪⫢ẘᛶ≀㉁ AFB1 ཪࡣ CPA ࢆ 24 㛫᭚㟢ᚋ㸪⣽⬊ࢆᅇࡋ RNA
ᢳฟ㸪GeneChip®Human Genome U133 Plus 2.0 Array ࢆ⏝࠸࡚⥙⨶ⓗ㑇ఏᏊⓎ⌧ኚືゎᯒࢆᐇࡋ
ࡓ㸬ᑐ↷ࡋ࡚㸪HepG2 ⣽⬊ࢆ⏝࠸ࡓ㸬✀ᚋ୍ᬌ᥋╔ࡉࡏࡓ HepG2 ⣽⬊ AFB1 ཪࡣ CPA ࢆ 24 㛫᭚㟢ᚋ㸪ྠᵝࡢ᪉ἲ࡛⥙⨶ⓗ㑇ఏᏊⓎ⌧ኚືゎᯒࢆᐇࡋࡓ㸬54675 probe set ࡢ㑇ఏᏊࢆ
ᑐ㇟ࡋࡓࡀ㸪ྜ≀㉳ᅉࡢኚື㑇ఏᏊᩘࢆTable 4-1 ♧ࡍ㸬⥆࠸࡚ AFB1 ཬࡧ CPA ࡢẘᛶⓎ
⌧㛵ࢃࡿࡇ࡛▱ࡽࢀࡿp53 㛵㐃㑇ఏᏊࡘ࠸࡚ᢳฟࡋ㸪ྜ≀᭚㟢ࡼࡿኚື⤖ᯝࢆ Spotfire
DecisionSite 9.1.1㸦TIBCO Software㸧ࢆ⏝࠸࡚㝵ᒙⓗࢡࣛࢫࢱࣜࣥࢢゎᯒࢆ⾜࠸㸪⤖ᯝࢆࣄ࣮ࢺ࣐ ࢵࣉ࡛♧ࡋࡓ㸦Fig.4-2㸧㸬
ࡑࡢ⤖ᯝ㸪AFB1 ཪࡣ CPA ฎ⌮ࡼࡿ Control ⩌ᑐࡍࡿኚື㑇ఏᏊᩘࡣ㸪2 ಸࡲࡓࡣ 0.5 ಸࢆ
threshold ࡋࡓሙྜ HepG2 ⣽⬊ẚ㍑ࡋ࡚ࡼࡾከࡃࡢ㑇ఏᏊࡀ HepaRG ⣽⬊࠾࠸࡚ኚືࡋ࡚࠸
ࡓ㸬AFB1㸦 ȝmol/L㸧ฎ⌮ࡘ࠸࡚ࡣ㸪2329 ಶࡢ㑇ఏᏊࡀ fold-change t2 ࡢኚື㸪1403 ಶࡢ
㑇ఏᏊࡀ fold-change 0.5 ࡢኚືࢆ HepaRG ⣽⬊࡛♧ࡋࡓ㸬HepG2 ⣽⬊࡛ࡣ㸪ྠ୍⃰ᗘࡢ AFB1
ࡢฎ⌮࠾࠸࡚㸪320 ಶࡢ㑇ఏᏊࡀ fold-changet 2 ࡢኚື㸪319 ಶࡢ㑇ఏᏊࡀ fold-change 0.5 ࡢኚ
ືࢆ♧ࡋࡓ㸬CPA㸦10 mmol/L㸧ฎ⌮ࡘ࠸࡚ࡣ㸪4424 ಶࡢ㑇ఏᏊࡀ fold-change t2 ࡢኚື㸪2275
ಶࡢ㑇ఏᏊࡀfold-change 0.5 ࡢኚືࢆ HepaRG ⣽⬊࡛♧ࡋࡓ㸬HepG2 ⣽⬊࡛ࡣ㸪ྠ୍⃰ᗘࡢ CPA
ࡢฎ⌮࠾࠸࡚㸪429 ಶࡢ㑇ఏᏊࡀ fold-change t2 ࡢኚື㸪518 ಶࡢ㑇ఏᏊࡀ fold-change 0.5 ࡢኚ
ືࢆ♧ࡋࡓ㸬p53 㛵㐃㑇ఏᏊࡘ࠸࡚ᢳฟࡋ࡚ゎᯒࡋࡓ⤖ᯝ࠾࠸࡚ࡶ㸪AFB1㸪CPA ඹ HepaRG
࠾࠸࡚㸪HepG2 ẚ㍑ࡋࡼࡾከࡃࡢ p53 㛵㐃ࡢ㑇ఏᏊࡢኚືࡀࡼࡾప࠸⃰ᗘ࡛ㄆࡵࡽࢀࡿ࠸
࠺⤖ᯝ࡛࠶ࡾ㸪AFB1 ཬࡧ CPA ࡢ᭚㟢ࡼࡾ㸪HepaRG ⣽⬊࡛ࡼࡾ p53 signaling ࡢࣃࢫ࢙࢘ 㸦Helton et al., 2007; Lukas et al., 2004㸧ࡀάᛶࡉࢀ࡚࠸ࡓ㸬ලయⓗኚືࡋ࡚࠸ࡿ㑇ఏᏊࡢࣉࣟ
࣮ࣈࢆTable 4-2 ᢤ⢋ࡋࡓࡀ㸪AFB1 ฎ⌮ࡼࡾ HepaRG ⣽⬊࡛ࡣ㸪G1 checkpoint 㛵㐃ࡍࡿ
CDKN1A (p21)㸪GADD45㸪FBXW7 ཬࡧ BTG2㸪Apoptosis signaling 㛵㐃ࡍࡿ BAX ཬࡧ APAF1㸪
Mismatch repair 㛵㐃ࡢ MSH2 ཬࡧ PCNA㸪Global genome repair 㛵ಀࡍࡿ XPC ཬࡧ DDB2㸪 Homologous recombination 㛵ࢃࡿ RAD51㸪p53 ࡢศゎไᚚ㛵ࢃࡿᢚไᅉᏊ࡛࠶ࡿ MDM2 ࡞
ࡢ㑇ఏᏊࡀ2 ಸ௨ୖࡢୖ᪼ࢆ♧ࡋࡓ㸬୍᪉ HepG2 ⣽⬊࡛ࡣ㸪BTG2㸪CDKN1A㸪MDM2㸪DDB2㸪
GADD45A ࡢࡳኚືࡀ☜ㄆࡉࢀࡓ㸬CPA ฎ⌮ࡘ࠸࡚ࡶྠᵝࡢഴྥ࡛㸪2 ಸ௨ୖࡢୖ᪼ࡢኚືࢆ
♧ࡋࡓࡶࡢࡣ㸪HepaRG ⣽⬊࡛ࡣ BTG2㸪CDKN1A㸪FBXW7㸪GADD45A㸪XPC㸪APAF1 ࡞ࡢ㑇
ఏᏊࡢኚືࡀ☜ㄆࡉࢀࡓࡀ㸪HepG2 ⣽⬊࡛ࡣ㸪ᢳฟࡋࡓ㑇ఏᏊࡢ࠺ࡕኚືࡢഴྥࡣㄆࡵࡽࢀࡓࡀ㸪
Table 4-1 Number of up-regulated or down-regulated genes in HepG2 and HepaRG cells after exposure to aflatoxin B1 or cyclophosphamide
Number of genes affected by AFB1 or CPA with a threshold fold-change or < 0.5 after 24 h treatment of HepaRG and HepG2 cells is shown. Gene probes with detection call of “P” or “M” were used for analysis.
Figure 4-2 Gene expression profiles in p53 signaling pathway in HepG2 and HepaRG cells treated with (a) aflatoxin B1 or (b) cyclophosphamide
Gene expression was visualized as a heatmap by using hierarchal clustering with Spotfire DecisionSite 9.1.1 (TIBCO) using UPGMA method. Gene probes whose detection call was “P” or “M” were used for
ȝ0 ȝ0 ȝ0 ȝ0 10 mM 20 mM 2 fold - 293 320 521 110 429 0.5 fold - 109 319 762 196 518 2 fold 1302 1480 2329 - 1632 4424 0.5 fold 735 1084 1403 - 1373 2275 HepG2 HepaRG
Compound AFB1 CPA
Concentration
0 1
-1
Table 4-2 Gene expression changes in p53 signaling pathway after exposure of HepG2 and HepaRG cells to aflatoxin B1 or cyclophosphamide
Fold-change in gene expression related to p53 signaling pathway in treatment groups relative to control group is shown. Red and blue values represent fold-changes t2 and <0.5, respectively. N.E., not evaluated, because detection call was “A” in one or more samples.
2.81 PM 5.63 PM 11.25 PM 1.41 PM 2.81 PM 5.63 PM 10 mM 20 mM 10 mM 20 mM
fold fold fold fold fold fold fold fold fold fold
204859_s_at APAF1 1.60 1.51 1.75 1.65 2.22 2.28 1.20 1.11 0.98 0.47 209903_s_at ATR 1.03 0.89 0.93 1.60 1.49 1.58 1.03 0.96 1.22 0.79 208478_s_at BAX 1.63 1.45 1.71 2.34 2.60 2.59 0.97 0.81 1.08 0.99 201236_s_at BTG2 3.37 3.54 5.28 2.89 2.92 3.18 NE NE 1.90 2.69 202284_s_at CDKN1A 4.44 5.62 8.12 3.07 3.66 3.37 1.16 1.43 2.56 3.44 203409_at DDB2 1.77 1.79 2.27 2.04 2.51 2.38 0.98 0.62 0.75 0.54 229419_at FBXW7 1.19 1.28 1.82 2.26 2.71 3.34 1.19 1.18 1.57 2.92 203725_at GADD45A 1.76 1.95 2.61 2.58 2.63 3.28 1.42 1.81 2.50 4.97 211040_x_at GTSE1 0.95 0.75 0.81 1.21 1.28 1.41 1.00 0.73 0.90 0.86 229711_s_at MDM2 1.94 2.21 2.53 2.34 3.02 3.07 1.46 1.69 1.67 1.54 202520_s_at MLH1 1.11 1.05 1.08 1.52 1.46 1.42 0.99 0.90 0.92 0.71 209421_at MSH2 0.94 0.85 0.68 2.54 2.85 3.21 1.13 0.91 NE NE 209361_s_at PCBP4 1.06 1.00 1.36 1.39 1.20 1.14 0.95 0.74 1.21 1.41 201202_at PCNA 0.99 1.25 1.11 2.33 2.51 2.59 0.99 0.93 1.48 1.21 205024_s_at RAD51 1.20 1.03 0.99 2.36 2.19 2.54 1.22 1.09 NE NE 201746_at TP53 1.05 1.01 0.99 1.67 1.63 1.57 0.97 1.07 1.15 1.35 205667_at WRN 0.72 0.82 0.53 1.26 1.01 0.98 1.30 1.43 1.22 1.59 209375_at XPC 1.28 1.49 1.69 1.88 2.24 2.88 0.93 1.11 1.70 2.58
Probe Set ID Gene
Symbol
HepG2 HepaRG HepG2 HepaRG
4.3
➨
3 ⠇ ᑠᣓ
ᮏ❶࡛ࡣ㸪ศࡉࡏࡓHepaRG ⣽⬊ࢆ⏝࠸࡚㸪⫢ẘᛶ≀㉁ AFB1 ཪࡣ CPA ࢆ᭚㟢ࡋ㸪⥙⨶ⓗ㑇
ఏᏊⓎ⌧ኚືゎᯒࢆᐇࡋ㸪ࡑࡢ㑇ఏᏊኚືࢆ HepG2 ⣽⬊ẚ㍑ࡋࡓ㸬ࡑࡢ⤖ᯝ㸪AFB1 ཬࡧ
CPA ࡢ᭚㟢ࡼࡾ㸪HepG2 ⣽⬊ẚ㍑ࡋ㸪HepaRG ⣽⬊࡛ࡼࡾከࡃࡢ㑇ఏᏊࡀኚືࡋ࡚࠸ࡓ㸬ࡑ
ࢀࡒࢀࡢ⫢ẘᛶⓎ⌧࣓࢝ࢽࢬ࣒㛵ಀࡍࡿࡇ࡛▱ࡽࢀࡿ p53 㛵㐃㑇ఏᏊࡘ࠸࡚ࡶ㸪HepaRG
⣽⬊࡛ࡼࡾከࡃ㑇ఏᏊࡀHepG2 ⣽⬊ẚ㍑ࡋኚືࡋ࡚࠸ࡓ㸬AFB1 ࡣ CYP1A2㸪CYP3A4 ࡞࡛
௦ㅰⓗάᛶࢆཷࡅ࡚⏕ᡂࡍࡿ AFB1 exo-8,9 epoxide యࡀẘᛶࢆ♧ࡍࡇ࡛▱ࡽࢀ࡚࠸ࡿ
㸦McLean et al., 1995; Gallagher et al., 1996㸧㸬CPA ࡣ CYP2B6㸪CYP3A4㸪CYP2C8/9 ࡞ࡼࡾ⏕
ᡂࡉࢀࡿ4-hydroxy CPA㸪ࡲࡓ᭱⤊ⓗ⏕ᡂࡍࡿ acrolein ཬࡧ phosphoramide mustard ࡞ࡼࡾẘ
ᛶࢆⓎ⌧ࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ㸦Chang et al., 1997; Gut et al., 2000; Tong et al., 2017㸧㸬ࡇࢀࡽࡢ
௦ㅰ≀ࡀDNA 㞀ᐖࢆᘬࡁ㉳ࡇࡍࡇ࡛㸪DNA damage response ࡛࠶ࡿ p53 㛵㐃㑇ఏᏊࡢኚືࡀᘬ
ࡁ㉳ࡇࡉࢀࡿ㸦Denissenko et al., 1999; Roy et al., 1999; Suzuki et al., 2008; Boehme et al., 2010㸧㸬 HepaRG ⣽⬊ࡀ HepG2 ẚ㍑ࡋ㸪⸆≀௦ㅰάᛶࡀ㧗࠸ࡇࡽ㸪ࡇࢀࡽࡢ DNA 㞀ᐖࢆᘬࡁ㉳ࡇ
ࡍ௦ㅰ≀ࡀHepaRG ⣽⬊࡛ࡼࡾከࡃ⏕ᡂࡉࢀ DNA 㞀ᐖࢆᘬࡁ㉳ࡇࡋ㸪ࡼࡾឤᗘࡼࡃ p53 㛵㐃㑇
5.
➨
5 ❶ ⫢ẘᛶ≀㉁ࡢ௦ㅰ≀ࡼࡿẘᛶⓎ⌧ホ౯
5.1
➨
1 ⠇ ⥴ゝ
⫢ẘᛶࡢⓎ⌧ࡣᵝࠎ࡞せᅉࡀ㛵ಀࡋ࡚࠸ࡿ⪃࠼ࡽࢀࡿࡀ㸪ࡑࡢ୰࡛ࡶ⫢⮚ࡢᶵ⬟ࡢ≉ᚩࡢ ୍ࡘ࡛࠶ࡿ⸆≀௦ㅰ㓝⣲ࡼࡾ㸪ྜ≀ࡀ⫢⮚࡛௦ㅰࢆ࠺ࡅ⏕ᡂࡉࢀࡿ௦ㅰ≀ࡀ⫢ẘᛶࡢⓎ⌧ 㛵ࡍࡿࡇࡶࡋࡤࡋࡤ࠶ࡿ㸬➨2 ❶ࡢ᳨ウࡽ㸪ศࡋࡓ HepaRG ⣽⬊ࡣ➨ I ┦࣭➨ II ┦௦ㅰ㓝 ⣲ࡶ☜ㄆࡋࡓศᏊ✀ࡘ࠸࡚ࡣάᛶࢆࣄࢺ⫢⣽⬊୪ࡳಖ᭷ࡋ࡚࠸ࡓ㸬ࡲࡓࡇࢀࡲ࡛ࡢ᳨ウ ࡽHepaRG ⣽⬊ࢆ⏝࠸࡚⫢ẘᛶ≀㉁ࡢẘᛶⓎ⌧ࢆホ౯࡛ࡁࡿ⣽⬊⣔࡛࠶ࡿࡇࡀ♧၀ࡉࢀࡓࡀ㸪 ᮏ❶࡛ࡣࡑࡢ⫢㞀ᐖࡢⓎ⌧ࡀ௦ㅰࢆࡋࡓẘᛶⓎ⌧࡛࠶ࡿ㸪௦ㅰ≀ࡢẘᛶⓎ⌧ࡢᙳ㡪ࢆホ౯ ࡛ࡁࡿヨ㦂⣔࡛࠶ࡿ࠸࠺ほⅬ࡛ヲ⣽࡞᳨ウࢆ⾜ࡗࡓ㸬5.2
➨
2 ⠇ ⣽⬊ẘᛶⓎ⌧ᑐࡍࡿ CYP ࡼࡿ௦ㅰࡢᙳ㡪
5.2.1 ➨1 㡯 ࣄࢺ⤖⫢⣽⬊ࢆ⏝࠸ࡓ⣽⬊ẘᛶホ౯ ་⸆ရࡢ௦ㅰ㛵ࢃࡿ࡞⸆≀௦ㅰ㓝⣲CYP ࡀᣲࡆࡽࢀࡿࡀ㸪ሗ࿌ࡼࡿ㸪௦ㅰࡼࡾࢡ ࣜࣛࣥࢫࢆ࠺ࡅࡿ་⸆ရࡢ࠺ࡕ⣙70%ࡀ CYP ࡛௦ㅰࡉࢀࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ㸦Yokoi., 2009㸧㸬 ࡑࡢࡼ࠺་⸆ရࡢ௦ㅰᐤࡢࡁ࠸ CYP ࡛ࡣ࠶ࡿࡀ㸪CYP ࡼࡗ࡚⏕ᡂࡍࡿ௦ㅰ≀ࡀ⫢ẘ ᛶࡢⓎ⌧㛵ࡋ࡚࠸ࡿሙྜࡶከ࠸㸬➨4 ❶㸪➨ 3 ⠇࡛㏙ࡓࡼ࠺㸪ᅇ⫢ẘᛶ≀㉁ࡋ࡚ ⏝ࡋࡓAFB1㸪CPA ࡞ࡶ CYP ࡼࡾ⏕ᡂࡍࡿ௦ㅰ≀ࡀࡑࡢẘᛶⓎ⌧㛵ࡋ࡚࠸ࡿࡇࡀ▱ࡽ ࢀ࡚࠸ࡿ㸬ᮏ⠇࡛ࡣCYP ࡼࡿ௦ㅰάᛶࡼࡾᘬࡁ㉳ࡇࡉࢀࡿ⣽⬊ẘᛶࡀ HepaRG ⣽⬊᳨࡛ฟ ࡛ࡁࡿࢆ᳨ウࡍࡿࡓࡵCYP ࡢ㜼ᐖࢆ⏝࠸࡚⣽⬊ẘᛶࡢⓎ⌧ࡢᙳ㡪ࢆホ౯ࡋࡓ㸬ࡲࡎࣄࢺ⤖⫢⣽⬊ࢆ⏝࠸࡚ AFB1 ཬࡧ CPA ࡼࡿ⣽⬊ẘᛶࡢ CYP ࡢ㜼ᐖࡢᙳ㡪ࡢホ౯
ࢆ⾜ࡗࡓ㸬ࣄࢺ⤖⫢⣽⬊ࡣ➨2 ❶㸪➨ 3 ❶࡛⏝ࡋࡓྠࡌࣟࢵࢺࡢࣄࢺ⤖⫢⣽⬊㸦Cryopreserved
Human Hepatocyte㸪Plateable, BioreclamationIVT ♫㸧ࢆ 3 ࣟࢵࢺ㸦3 ࢻࢼ࣮㸧⏝ࡋࡓ㸬CYP ࡢ㜼
ᐖࡋ࡚ࡣ㠀≉␗ⓗ࡞CYP ࡢ㜼ᐖ࡛࠶ࡿ ABT ࢆ⏝࠸ࡓ㸬ABT ࡣ Mechanism-based inactivator
ࡋ࡚▱ࡽࢀ㸪⮬㌟ࡀ CYP ࡛௦ㅰࡉࢀ CYP ࡢάᛶ୰ᚰࡢ࣒࣊ࢆ N ࣝ࢟ࣝࡍࡿࡇ࡛ CYP
ࢆศᏊ✀㠀≉␗ⓗ㜼ᐖࡍࡿ㜼ᐖ࡛࠶ࡿ㸦Fig. 5-1㸧㸦Ortiz et al., 1981, Paul et al., 1984, and Emoto
et al., 2005㸧㸬ᮏ᳨ウ࡛ࡣࣄࢺ⤖⫢⣽⬊ AFB1 CPA ྠ ABT㸦500 Pmol/L㸧ࢆ᭚㟢ࡋ㸪
48 㛫ᚋ⣽⬊㞀ᐖᛶࢆホ౯ࡋࡓ㸬࡞࠾㸪ᮏ ABT ࡢ᮲௳ࡣ᪤ሗ࿌㸦Ogimura et al., 2011㸧࡛⏝ ࡉࢀࡓ᮲௳ࢆ⏝࠸ࡓࡀ㸪CYP ࡀ༑ศ㜼ᐖࡉࢀࡿࡇࢆ☜ㄆࡋࡓ㸦Fig. 5-2㸧㸬⣽⬊㞀ᐖᛶࡢᣦᶆ
ࡣATP ࢵࢭࢆ⏝࠸ࡓ㸬
ࡑࡢ⤖ᯝ㸪AFB1 ࡣࡍ࡚ࡢࣟࢵࢺࡢࣄࢺ⤖⫢⣽⬊⣽⬊ẘᛶࢆ♧ࡋࡓࡀ㸪CYP ࡢ㜼ᐖ࡛
࠶ࡿ ABT ࢆඹฎ⌮ࡍࡿࡇࡼࡗ࡚㸪ࡑࡢ⣽⬊ẘᛶࡣῶᙅࡋ㸪㜼ᐖࡉࢀࡓ㸬ࡲࡓ㸪CPA
ࡢ⣽⬊ẘᛶࡀㄆࡵࡽࢀࡓࣟࢵࢺYEM ࡘ࠸࡚ࡣ㸪CPA ࡼࡗ࡚ᘬࡁ㉳ࡇࡉࢀࡓ⣽⬊ẘᛶࡀ ABT
ࡼࡾ᭷ពῶᙅࡋ࡚࠸ࡿࡇࡀ☜ㄆࡉࢀ㸪AFB1 ཬࡧ CPA ࡢࣄࢺ⤖⫢⣽⬊ࡢẘᛶࡣ CYP ࡼࡿ௦ㅰ≀ࡀᐤࡋ࡚࠸ࡿࡇࡀ♧၀ࡉࢀࡓ㸦Fig. 5-3㸧㸬
Figure 5-1 Structure of 1-aminobenzotriazole (ABT), a non-specific broad CYP Inhibitor
N N N
Figure 5-2 Inhibitory Effects of ABT against CYP activity in cryopreserved human hepatocytes Inhibitory effects of 500 Pmol/L ABT on several CYP activities (CYP1A2; phenacetin O-deethylase,
0.0 1.0 2.0 3.0 4.0 5.0
lot SHM lot CPQ lot YEM
(p m ol· m in -1·m g pro tei n -1) CYP2C9 activity $%7 $%7 0.0 2.0 4.0 6.0
lot SHM lot CPQ lot YEM
(p m ol· m in -1·m g pro tei n -1) CYP1A2 activity $%7 $%7 ND ND ND 0.0 1.0 2.0 3.0 4.0
lot SHM lot CPQ lot YEM
(p m ol· m in -1·m g pro tei n -1) CYP2C19 activity $%7 $%7 ND/ND ND 0.0 0.3 0.6 0.9 1.2
lot SHM lot CPQ lot YEM
(p m ol· m in -1·m g pro tei n -1) CYP2D6 activity $%7 $%7 0 20 40 60 80 100
lot SHM lot CPQ lot YEM
(p m ol· m in -1·m g pro tei n -1) CYP3A4 activity $%7 $%7 0.349/0.062
Figure 5-3 Effects of broad CYP inhibitor on cytotoxicity of AFB1 and CPA in cryopreserved human hepatocytes
Cytotoxicity of AFB1 and CPA was examined in cryopreserved human hepatocytes (CHHs) with or without 1-aminobenzotriazole (ABT), a non-selective CYP inhibitor. Results are black bar for with ABT and white bar for without ABT. CHHs (Lot. SHM; (a), CPQ; (b), and YEM; (c)) were exposed to AFB1 (-1) and CPA (-2) for 48 h with or without ABT (ABT(-) or ABT(+)). ABT concentrations were 500 Pmol/L. Cell viability was evaluated using CellTiter-GloTM luminescent cell viability assay. Results are
normalized to control cells and expressed as means r S.D. (n 3 cultures). The data shown in Figure 5-3
0 20 40 60 80 100 120 140 0 0.2 0.6 2 6 20 C ell v ia b ilit y (% o f co n tr ol) Concentration (Pmol/L) Aflatoxin B1 ABT (-) ABT (+) (a)-1 0 20 40 60 80 100 120 140 0 250 750 2500 7500 25000 C ell v ia b ilit y (% o f co n tr ol) Concentration (Pmol/L) Cyclophosphamide ABT (-) ABT (+) (a)-2 0 20 40 60 80 100 120 140 0 0.6 2 6 20 C ell v ia b ilit y (% o f co n tr ol) Concentration (Pmol/L) Aflatoxin B1 ABT (-) ABT (+) (b)-1 0 20 40 60 80 100 120 140 0 750 2500 7500 25000 C ell v ia b ilit y (% o f co n tr ol) Concentration (Pmol/L) Cyclophosphamide ABT (-) ABT (+) (b)-2 0 20 40 60 80 100 120 140 0 0.6 2 6 20 C ell v ia b ilit y (% o f co n tr ol) Concentration (Pmol/L) Aflatoxin B1 ABT (-) ABT (+) (c)-1 0 20 40 60 80 100 120 140 0 750 2500 7500 25000 C ell v ia b ilit y (% o f co n tr ol) Concentration (Pmol/L) Cyclophosphamide ABT (-) ABT (+) (c)-2 *** *** *** *** *** *** ** ** *** *** ** * *** *** *
5.2.2 ➨2 㡯 HepaRG ⣽⬊ࢆ⏝࠸ࡓ⣽⬊ẘᛶホ౯
➨1 㡯࡚ࣄࢺ⤖⫢⣽⬊ࢆ⏝࠸࡚㸪CYP ࡼࡿ௦ㅰ≀ࡼࡿẘᛶⓎ⌧ࡢᙳ㡪ࢆ☜ㄆࡍࡿࡇ
ࡀ࡛ࡁࡓࡀ㸪HepaRG ⣽⬊࡛ྠᵝ CYP ࡼࡿ௦ㅰⓗάᛶࡼࡿẘᛶⓎ⌧ࡢᙳ㡪ࢆホ౯࡛ࡁ
ࡿ᳨ウࢆ⾜ࡗࡓ㸬⫢ẘᛶ≀㉁ࡋ࡚ࣄࢺ⤖⫢⣽⬊ࡢ᳨ウྠᵝAFB1㸪CPA ࢆ⏝࠸࡚㸪
HepaRG ⣽⬊ࡢ⣽⬊ẘᛶᑐࡍࡿ ABT ࡢඹฎ⌮ࡼࡿᙳ㡪ࢆホ౯ࡋࡓ㸬ABT ࡢฎ⌮᮲௳ࡢ᳨
ウࡋ࡚๓⃰ᗘࢆࡩࡗ࡚⣽⬊ẘᛶཬࡧCYP άᛶࡢᙳ㡪ࢆホ౯ࡋ㸪᮲௳ࢆタᐃࡋࡓ㸬⣽⬊ẘ
ᛶࡢホ౯ࡋ࡚ࡣATP ࢵࢭ㸪LDH ࢵࢭ㸦LDH Cytotoxicity Detection Kit㸪Takara㸧ࢆ⏝࠸
ࡓ㸬ࡑࡢ⤖ᯝABT ࡣ HepaRG ⣽⬊ᑐࡋ࡚㸪100㹼500Pmol/L ࡢ⃰ᗘ⠊ᅖ࠾࠸࡚᫂ࡽ࡞⣽⬊
ẘᛶࡣ♧ࡉ࡞ࡗࡓ㸦Fig. 5-4 (a)-1, (a)-2㸧㸬⥆࠸࡚㸪ABT ࡢ⸆≀௦ㅰ㓝⣲άᛶᑐࡍࡿ㜼ᐖຠᯝ
ࡶ☜ㄆࡋࡓ㸬⃰ᗘࢆ100 Pmol/L ཬࡧ 500 Pmol/L ࡢ 2 ⃰ᗘ㸪๓ฎ⌮ࡢ᭷↓ࡢ᳨ウ࠸࠺ࡇ࡛㓝
⣲άᛶホ౯ࡢࡓࡵࡢᇶ㉁᭚㟢ࡢ1 㛫๓ࡢ ABT ࡢ᭚㟢ࡢ᭷↓ࡢ 4 ᮲௳㸦ABT 100 Pmol/L ๓ฎ⌮
࡞ࡋ㸭ABT 500 Pmol/L ๓ฎ⌮࡞ࡋ㸭ABT 100 Pmol/L ๓ฎ⌮ 1 㛫㸭ABT 500 Pmol/L ๓ฎ⌮ 1
㛫㸧ࡘ࠸࡚㸪CYP6 ศᏊ✀㸪ᢪྜ㓝⣲ 2 ศᏊ✀ࡘ࠸࡚ホ౯ࢆ⾜ࡗࡓ㸬࡞࠾ࡇࡢࡁࡢ HepaRG
⣽⬊ࡢศ᮲௳ࡣ㸪ᅇヨ㦂⣔⏝࠸ࡓ7 ᪥㛫ศᚋࡢ HepaRG ⣽⬊ࡢάᛶࡼࡾࡶ㸪㓝⣲άᛶࡀ
㧗࠸㸪ศDay9㸪Day14㸪Day16 ࡢ HepaRG ⣽⬊ࢆ⏝࠸࡚᮲௳ࢆ᳨ウࡋࡓ㸬ࡑࡢ⤖ᯝ㸪ࡍ࡚ࡢ
CYP ศᏊ✀ࡘ࠸࡚ ABT ࡢ㜼ᐖຠᯝࡀㄆࡵࡽࢀࡓ㸦Fig. 5-5㸧㸬CYP2B6㸪CYP2C19㸪CYP3A4
ࡘ࠸࡚ࡣ㸪⃰ᗘࡀ㧗࠸᮲௳ࡢ㜼ᐖຠᯝࡀ㧗ࡃ㸪๓ฎ⌮ࡢຠᯝࡶㄆࡵࡽࢀࡓࡀ㸪100Pmol/L ๓ฎ⌮
࡞ࡋࡢ᮲௳࡛ࡶ㜼ᐖຠᯝࡣ༑ศ࡛࠶ࡗࡓ㸬CYP2C9 ࡘ࠸࡚ࡶ⃰ᗘࡀ㧗࠸᪉ࡀࡼࡾ㜼ᐖຠᯝࡀ㧗 ࠸ഴྥ࡛࠶ࡗࡓࡀ㸪యⓗ㜼ᐖຠᯝࡀᙅࡃ㸪ࡇࢀࡣ᪤ሗ㸦Emoto et al., 2003㸧ࡢ㏻ࡾ࡛࠶ࡗࡓ㸬 CYP1A2, CYP2D6 ࡘ࠸࡚ࡣࡢ᮲௳࡛ࡶ༑ศ࡞㜼ᐖຠᯝࡀㄆࡵࡽࢀࡓ㸬UGT ཬࡧ SULT ࡘ࠸
࡚ࡣ㸪㜼ᐖຠᯝࡣㄆࡵࡽࢀ࡞ࡗࡓ㸬ࡇࢀࡽࡢ᳨ウ⤖ᯝࡽ㸪CYP2C9 ࡢ㜼ᐖຠᯝࡣᙅ࠸ࡶࡢࡢ㸪
ABT 100 Pmol/L ࡲࡓࡣ 500 Pmol/L ࡛ࡶࡰྠ⛬ᗘࡢ㜼ᐖ࡛࠶ࡗࡓࡇ㸪100 Pmol/L ࡢ᮲௳࡛
ࡶ༑ศ࡞CYP άᛶࡢ㜼ᐖຠᯝࡀㄆࡵࡽࢀࡓࡓࡵ㸪ࡼࡾ⣽⬊ࡢᙳ㡪ࡀᑡ࡞࠸⃰ᗘ࡛㸪ࡲࡓ㸪ᐇ㦂
ୖࡢ᧯సᛶࡶ⪃៖ࡋ㸪ᮏヨ㦂⣔࡛ࡢHepaRG ⣽⬊ᑐࡍࡿ ABT ࡢฎ⌮᮲௳ࡣ⃰ᗘ 100Pmol/L㸦๓
ฎ⌮↓ࡋ㸧ࡋࡓ㸬࡞࠾Ỵᐃࡋࡓ᮲௳࡛ศDay7 ࡢ HepaRG ⣽⬊ࢆ⏝࠸࡚㸪⤒ⓗ࡞ ABT ࡢ㜼
ᐖຠᯝࡢホ౯ࢆ⾜ࡗࡓ㸬ࡑࡢ⤖ᯝ㸪☜ㄆࡋࡓࡍ࡚ࡢCYP ศᏊ✀ࡘ࠸࡚㸪ABT ࡼࡿ㜼ᐖຠ
ᯝࡀㄆࡵࡽࢀ㸪ฎ⌮ᚋ 72 㛫ࡲ࡛㜼ᐖࡀ⥔ᣢࡉࢀ࡚࠾ࡾ㸪༑ศ࡞㜼ᐖ᮲௳࡛࠶ࡿࡇࡀ☜ㄆ࡛
ࡁࡓ㸦Fig. 5-6㸧㸬CYP2C9 ᑐࡍࡿ㜼ᐖຠᯝࡣࡢศᏊ✀ࡼࡾࡣపࡗࡓࡀ㸪㜼ᐖస⏝ࡣ 72 㛫 ࡲ࡛⥔ᣢࡋ࡚࠸ࡓ㸬
Figure 5-4 Cytotoxicity of ABT for 72 h exposure in HepaRG cells
Cytotoxicity of 1-aminobenzotriazole (ABT) was examined in HepaRG cells to find the dose concentration for treatment. HepaRG cells were exposed to ABT for 72 h and cytotoxicity assay were evaluated using both ATP assay ((a)-1) and LDH assay ((a)-2). ATP assay was conducted using CellTiter-GloTM
luminescent cell viability assay (n 3 cultures). LDH assay was conducted using LDH Cytotoxicity Detection Kit (n 3 cultures).
0 25 50 75 100 125 0 100 200 300 400 500 Ce ll v ia b ilit y ( % of c ont rol )
ABT concentration (Pmol/L)
0 20 40 60 80 100 0 100 200 300 400 500 LDH ( U /L)
ABT concentration (PPmol/L)
Figure 5-5 Inhibitory effects of ABT against drug-metabolizing enzyme activity in HepaRG cells
0 5 10 15
Day9 Day14 Day16
(p mo l· mi n -1·m g p rote in -1) CYP1A2 activity
ABT (-) ABT100 ABT500
ABT100 Pre1h ABT500 Pre1h
ND/ND/ND/ND ND/ND/ND/ND ND/ND/ND/ND 0 5 10 15 20 25 30
Day9 Day14 Day16
(p mo l· mi n -1·m g p rote in -1) CYP2B6 activity
ABT (-) ABT100 ABT500
ABT100 Pre1h ABT500 Pre1h
ND ND ND 0.0 1.0 2.0 3.0 4.0 5.0
Day9 Day14 Day16
(p mo l· mi n -1·m g p rote in -1) CYP2C9 activity
ABT (-) ABT100 ABT500
ABT100 Pre1h ABT500 Pre1h
0.0 2.0 4.0 6.0 8.0
Day9 Day14 Day16
(p mo l· mi n -1·m g p rote in -1) CYP2C19 activity
ABT (-) ABT100 ABT500
ABT100 Pre1h ABT500 Pre1h
ND ND ND 0.0 1.0 2.0 3.0 4.0 5.0
Day9 Day14 Day16
(p mo l· mi n -1·m g p rote in -1) CYP2D6 activity
ABT (-) ABT100 ABT500
ABT100 Pre1h ABT500 Pre1h
0 50 100 150 200 250 300
Day9 Day14 Day16
(p mo l· mi n -1·m g p rote in -1) CYP3A4 activity
ABT (-) ABT100 ABT500
ABT100 Pre1h ABT500 Pre1h
9.64/2.61/2.24/1.12 7.88/2.92/2.30/0.78 8.17/2.62/2.03/0.71 0 500 1000 1500 2000 2500
Day9 Day14 Day16
(p mo l· mi n -1·m g p rote in -1) UGT activity
ABT (-) ABT100 ABT500
ABT100 Pre1h ABT500 Pre1h
0 5 10 15 20 25 30
Day9 Day14 Day16
(p mo l· mi n -1·m g p rote in -1) SULT activity
ABT (-) ABT100 ABT500
Figure 5-6 Inhibitory effects of ABT (100Pmol/L) against CYP activity for 72 h in HepaRG cells Inhibitory effects of 100Pmol/L ABT on several CYP activities (CYP1A2; phenacetin O-deethylase, &<3& GLFORIHQDFމ-hydroxylase, CYP2C19; S-PHSKHQ\WRLQމ-hydroxylase, CYP2D6; bufuralol މ-K\GUR[\ODVH&<3$ PLGD]RODPމ-hydroxylase) in HepaRG cells were confirmed. Results are means r S.D (n 3). ABT(-): without ABT, ABT(+): with 100 Pmol/L ABT, ND: not detectable.
0 5 10 15 20 0hr 6hr 48hr 72hr (p m ol· m in -1·m g pro tei n -1)
CYP1A2 activity
$%7 $%7 ND ND ND 0.299 0 10 20 30 40 50 0hr 6hr 48hr 72hr (p m ol· m in -1·m g pro tei n -1)CYP2B6 activity
$%7 $%7 0.280 0.677 0 10 20 30 40 0hr 6hr 48hr 72hr (p m ol· m in -1·m g pro tei n -1)CYP2C9 activity
$%7 $%7 0 2 4 6 8 10 0hr 6hr 48hr 72hr (p m ol· m in -1·m g pro tei n -1)CYP2C19 activity
$%7 $%7 0.0 2.0 4.0 6.0 0hr 6hr 48hr 72hr (p m ol· m in -1·m g pro tei n -1)CYP2D6 activity
$%7 $%7 0.177 0.085 0.070 0.095 0 50 100 150 200 0hr 6hr 48hr 72hr (p m ol· m in -1·m g pro tei n -1)CYP3A4 activity
$%7 $%7 0.582 0.261 0.384ࡇࡢࡼ࠺タᐃࡋࡓABT ࡢ᮲௳ࢆ⏝࠸࡚㸪⣽⬊ẘᛶホ౯ࢆ࠾ࡇ࡞ࡗࡓ㸬ศࡉࡏࡓ HepaRG ⣽
⬊AFB1 CPA ྠ ABT㸦100Pmol/L㸧ࢆ᭚㟢ࡋ㸪48 㛫ᚋ⣽⬊㞀ᐖᛶࢆホ౯ࡋࡓ㸦ATP
ࢵࢭ㸧㸬ࡑࡢ⤖ᯝ㸪⃰ᗘ౫Ꮡⓗ࡞⣽⬊ẘᛶࢆ♧ࡋࡓ AFB1 ᑐࡋ࡚㸪ABT ฎ⌮࡛⣽⬊ẘᛶࡢ
᫂ࡽ࡞ῶᙅࡀㄆࡵࡽࢀࡓ㸦Fig. 5-7, (a)-1㸧㸬ࡲࡓ᭱㧗⃰ᗘࡢ 25000 Pmol/L ࡛⣽⬊ẘᛶࢆ♧ࡋࡓ
CPA ࡘ࠸࡚ࡶ㸪ABT ࡢฎ⌮࡛㸪25000Pmol/L ࡢ⣽⬊ẘᛶࡀⴭࡋࡃῶᙅࡋ࡚࠸ࡓ㸦Fig. 5-7, (a)-2㸧㸬
ࡇࢀࡽࡢ⤖ᯝࡽ㸪CYP ࡼࡗ࡚⏕ᡂࡉࢀࡿ௦ㅰ≀ࡼࡿẘᛶࢆ HepaRG ⣽⬊࡛ࡽ࠼ࡽࢀ࡚࠸
ࡿࡇࡀ♧၀ࡉࢀࡓ㸬
Figure 5-7 Effects of broad CYP inhibitor on cytotoxicity of AFB1 and CPA in HepaRG cells Cytotoxicity of AFB1 and CPA was examined in HepaRG cells with or without 1-aminobenzotriazole (ABT), a non-selective CYP inhibitor. Results are white bar for without ABT and black bar for with ABT. HepaRG cells were exposed to AFB1 (-1) and CPA (-2) for 48 h with or without ABT (ABT(-) or ABT(+)). ABT concentrations were 100 Pmol/L. Cell viability was evaluated using CellTiter-GloTMluminescent cell
viability assay. Results are normalized to control cells and expressed as means r S.D. (n 3 cultures). The data shown in Figure 5-7 (without ABT) and Figure 3 (a, b) are the same. Student's t-tests were conducted at the same concentration of test compound with or without ABT (*:p< 0.05, **:p< 0.01, ***p< 0.001).
0 20 40 60 80 100 120 0 0.2 0.6 2 6 20 C ell vi ab ilit y (% o f c o n tr o l) Concentration (Pmol/L)
