臨床応用を指向した1点採血による血中6β-hydroxycortisol/cortisol 濃度比を用いたヒト in vivo CYP3A 活性評価法の開発と応用

(1)

1 6β-hydroxycortisol/cortisol

in vivo CYP3A

In vivo CYP3A phenotyping with a single blood sample

for personalized medicine as probed

by plasma 6β-hydroxycortisol/cortisol ratio

(2)

(3)

1.3.1.2 6β-hydroxycortisol 12 1.3.1.3 14 1.3.1.4 HPLC 15 1.3.1.5 16 1.3.1.6 18 1.3.1.7 18 1.3.1.8 19 1.3.2 6β-hydroxycortisol cortisol ( ) 19 1.3.2.1 6β-hydroxycortisol cortisol 19 1.3.2.2 HPLC 21 1.3.2.3 23 1.3.2.4 24 1.3.2.5 25 1.3.2.6 25 1.3.3 6β-hydroxycortisol cortisol 26 1.3.3.1 26 1.3.3.2 6β-OHF cortisol 27

2 6β-hydroxycortisol /cortisol CYP3A

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2.3 32 2.3.1 6β-hydroxycortisol/cortisol CYP3A 32 2.3.1.1 6β-hydroxycortisol cortisol 34 2.3.1.2 6β-hydroxycortisol/cortisol 36 2.3.1.3 CLm(6β) 6β-hydroxycortisol/cortisol 39 2.3.2 6β-hydroxycortisol/cortisol 42 2.3.2.1 6β-hydroxycortisol/cortisol 42 2.3.2.2 6β-hydroxycortisol/cortisol 44 2.3.3 6β-hydroxycortisol/cortisol CYP3A 45 3 Dried blood spots 6β-hydroxycortisol/cortisol

CYP3A

3.1 48

3.2 49

3.2.1 49

3.2.2 LC-MS/MS 49

3.2.3 Dried blood spots 49

3.2.4 49

3.2.5 50

3.2.6 50

3.2.7 6β-hydroxycortisol cortisol 50

3.2.8 Dried blood spots 6β-hydroxycortisol/cortisol in

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3.3.7 , 6β-hydroxycortisol cortisol 57 3.3.8 Dried blood spots 6β-hydroxycortisol/cortisol in

vivo CYP3A 58

61 65

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1

( ) ( ) 2012

5 .

(Therapeutic drug monitoring: TDM)

P450 (CYP) 500

1 80% 1) _CYP

CYP1A2, CYP2D6, CYP2C19, CYP3A

CYP 95% 1) _CYP3A 5 ~ 20 2-4) _CYP3A HMG-CoA CYP3A 5) 6) CYP3A CYP3A CYP3A

CYP3A CYP3A4, CYP3A5, CYP3A7, CYP3A43

CYP3A4, CYP3A5 CYP3A4 CYP 30%

CYP3A5 CYP3A4 4,7,8) _CYP3A7

CYP3A4 4,7) _CYP3A43

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2

CYP3A4 CYP3A4*20 CYP3A4*8, *11, *13, *16,

*17, *22 10) _CYP3A5 _CYP3A5*3

10,11)

CYP3A

Gold standard (Food

and drug administration: FDA) (European medicines agency: EMA) 12)

1 ~ 3 mg 1

~ 3 µg 13) ₄ _CYP3A

14)

CYP3A

quinine/3-hydroxyquinine 15) _{omeprazole/omeprazole sulphone}

16) ₁

cholesterol CYP3A 4β-hydroxycholesterol

2008 Diczfalusy 17) _CYP3A 4β-hydroxycholesterol 4β-hydroxycholesterol/cholesterol 18) 19) 4β-hydroxycholesterol 17 20) ₂ 21,22) _CYP3A

cortisol CYP3A 6β-hydroxycortisol (6β-OHF)

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3 CYP3A CYP3A 1 6β-OHF/cortisol in vivo ( ) 7) _{6β-OHF/cortisol} _CYP3A

6β-OHF/cortisol cortisol 6β-OHF CLm(6β)

6β-OHF/cortisol Shin 27) _Woolsey 28)

1 cortisol

CYP3A 6β-OHF cortisol 1% 29)

6β-OHF/cortisol CYP3A 6β-OHF cortisol 2025 30% (2025 ) TDM CYP3A 2014 4 HbA1c

(Dried blood spots: DBS) CYP3A

DBS 1960 Robert Guthrie

30) _DBS

DBS DBS

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4

1 LC-MS/MS

6β-OHF cortisol (

) Electrospray ionization negative ion

mode (ESI ( )) 6β-OHF cortisol

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5

1 6β-hydroxycortisol cortisol LC-MS/MS 1.1

6β-hydroxycortisol (6β-OHF) cortisol CYP3A

6β-OHF/cortisol CYP3A

6β-OHF HPLC

LC-MS/MS 31-33) _HPLC

methylprednisolone beclomethasone 6β-OHF cortisol

31) _{6α-hydroxycholesterol} _6β-OHF

6α-OHF 32) _GC-MS

6β-OHF 33) _cortisol ₁₀

~ 250 ng/mL 34)

6β-OHF cortisol 6β-OHF cortisol 1% 29)

6β-OHF

6β-OHF 6β-OHF/cortisol

6β-OHF cortisol

Zhiri 35) _6β-OHF _6β-OHF

0.30 ~ 1.99 ng/mL LC-MS/MS

Imamura 36,37) Organic anion transporter 3 (OAT3)

(11)

6 )

6β-OHF cortisol LC-MS/MS ( )

1.2 1.2.1

6β-hydroxycortisol (6β,11β,17α,21-tetrahydroxypregen-4-ene-3,20-dione: 6β-OHF) Steraloids (Wilton, NH, USA) cortisol Sigma-Aldrich (St. Louis, MO,

USA) [1,2,4,19-13_C 4]cortisol (cortisol-13_C 4) 47) cortisol-13C4 97.3atom%, 95.8% 6β-[9,11,12,12-2_H 4]hydroxycortisol (6β-OHF-2_H 4) Sigma-Aldrich 6β-OHF-2H4 98% LC-MS (Tokyo, Japan) (Osaka, Japan) , ( )

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7

1.2.4 6β-hydroxycortisol cortisol (

) 1.2.4.1 LC-MS/MS

LC-MS/MS Agilent 1200 system (Agilent, Santa Clara. CA, USA) Agilent 6410 triple quadropole mass spectrometer

Phenomenex (CA, USA) Synergi 4u MAX-RP 80 analytical column (250 × 2.0 mm I.D., 4.0 µm), Security Guard Cartridges C12 (4 × 2.0 mm I.D.)

35 0.2 mL/min 0.2% 0.01 M

20% (0–4 min), 65% (4.1–7 min), 50% (7.1–9 min), 90% (9.1–10 min), 75% (10.2–13 min), 20% (13.2–25

min) 25 Electrospray ionization (ESI)

Nebulizer gas (10 L/min) Collision gas (50 psi, 350 ) Multiple reaction monitoring (MRM) Positive ion mode

Precursor ion Product ion 6β-OHF m/z 484 78

6β-OHF-2_H

4 m/z 488 78 cortisol m/z 468 78, cortisol-13C4 m/z 472 78 Collision energy (C.E.) 55 eV

1.2.4.2

1 mL 6β-OHF-2_H

4 (1.490 ng) cortisol-13C4 (54.72 ng) Sep-Pak C18 Plus short-body cartridge (Waters, Milford, MA, USA)

(2 mL × 3 ) 5 mL

Sep-Pak C18 Plus short-body cartridge 8 mL ,

4 mL

1.2.4.3

(50 µL)

(50 µL) 10

(20 mg) 2-methyl-6-nitrobenzoic anhydride (20 mg) 4-dimethylaminopyridine

(10 mg) (1 mL) 1% (1 mL)

(2 mL × 3 )

5 mL Sep-Pak C18 Plus short-body cartridge 8 mL

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8

(100 µL) (Pall, NY, USA)

2 µL LC-MS/MS 1.2.4.4

Signal to noise ratio (S/N) > 3 (n = 6)

S/N > 10 6β-OHF (38.51 pg/mL) cortisol (38.10 pg/mL) 1.2.4.5 5 (6β-OHF: 0.411 ~ 2.465 ng/mL, cortisol: 20.22 ~ 252.75 ng/mL) LC-MS/MS 4 7 14 1.2.4.6

6β-OHF cortisol 6β-OHF (0.082, 0.411, 0.822, 1.232, 1.643, 2.465 ng) cortisol (20.22, 60.66, 101.10, 141.54, 181.98, 252.75 ng) 6

6β-OHF-2_H

4 (1.490 ng) cortisol-13C4 (54.72 ng) 1.2.4.3

LC-MS/MS 1.2.4.1 Precursor ion Product ion

1.2.4.7

1 mL (n = 18) 3 1 (n = 6) 2

6β-OHF cortisol 6β-OHF-2_H

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9

1.2.5 6β-hydroxycortisol cortisol (

) 1.2.5.1 LC-MS/MS

LC-MS/MS LC/MS 8030 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan) LC-20AD binary gradient pump, SIP-20A auto-sampler, DGU-20A3 degasser,

CTO-20AC column oven LC system

Phenomenex (CA, USA) Kinetex 2.6u C8 100 column (75 × 3.0 mm I.D., 2.6 µm), Security Guard ULTRA Cartridge C8 (3.0 mm I.D.) 40

0.45 mL/min 0.4%

20% (0 min), 90% (9–10 min), 20% (10.5–15 min)

15 ESI Nebulizer gas (3 L/min)

Drying gas (15 L/min) Collision voltage 6 kV, Source temperature 400 , Desolvation temperature 290 MRM Negative ion mode Precursor ion Product ion 6β-OHF m/z 423 347 6β-OHF-2_H

4

m/z 427 351 cortisol m/z 407 331, cortisol-13_C

4 m/z 411 335

C.E. 6β-OHF 6β-OHF-2_H

4 19 eV, cortisol cortisol-13C4 15 eV

1.2.5.2

1 mL 6β-OHF-2_H

4 (1.490 ng) cortisol-13C4 (54.72 ng) Sep-Pak C18 Plus short-body cartridge (Waters, Milford, MA, USA)

(2 mL × 3 ) 5 mL

Sep-Pak C18 Plus short-body cartridge 8 mL ,

4 mL (75 µL)

20 µL LC-MS/MS 1.2.5.3

S/N > 3 (n = 6) S/N > 10

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10 1.2.5.4 5 mL (n = 3) 1 mL 1.2.5.2 LC-MS/MS 20 21 48 1 mL 1.2.5.5

6β-OHF cortisol 6β-OHF (0.082, 0.411, 0.822, 1.232, 1.643, 2.465 ng) cortisol (20.22, 60.66, 101.10, 141.54, 181.98, 252.75 ng) 6

6β-OHF-2_H

4 (1.490 ng) cortisol-13C4 (54.72 ng)

(75 µL) LC-MS/MS

1.2.5.1 Precursor ion Product ion

1.2.5.6

1 mL (n = 18) 3 1 (n = 6) 2

6β-OHF cortisol 6β-OHF-2_H

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11 1.3 cortisol HPLC , GC-MS LC-MS 6β-hydroxycortisol (6β-OHF) 31-33)_, 6β-OHF cortisol 6β-OHF 1.3.1 6β-hydroxycortisol cortisol ( ) 1.3.1.1 LC-MS/MS LC-MS/MS 2-fluoro-1-methylpyridinium 40,41) 2-hydrazino-1-methylpyridine 40,42,43) _p- 40,44) 45,46) testosterone tetrahydrocortisol LC-MS/MS 45,46) _Yamashita 46)

cortisol Electrospray ionization positive ion mode (ESI (+)) [M + H]+ _{Precursor ion} _{m/z 124, 106, 78}

Product ion cortisol ESI (+) 5 ~ 10

6β-OHF Yamashita 48)

6β-OHF cortisol 6β-OHF cortisol

LC-MS/MS

Yamashita 45) _cortisol

cortisone A tetrahydrocortisol, allotetrahydrocortisol,

tetrahydrocortisone

(50 µL) (20 mg),

2-methyl-6-nitro benzoic anhydride (20 mg) 4-dimethylaminopyridine (10 mg)

(1 mL) (50 µL) (10

µL) 45 1% (1 mL)

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12 4-dimethylaminopyridine

6β-OHF 1 ng/mL (0.0027 nmol/mL)

4 6β-OHF 4

0.0106 nmol 1.31 ng

cortisol 10 ~ 250 ng/mL (0.028 ~ 0.691 nmol/mL) Yamashita 45)

1 1 mL 4.78 ~

86.99 ng Yamashita 300 ~ 4000

Yamashita 6β-OHF 0.82 ng LC-MS/MS Multiple reaction

monitoring (MRM) (R.S.D.)

33% ~ 44% (5 mg), 2-methyl-6-nitro benzoic

anhydride (5 mg), 4-dimethylaminopyridine (2.5 mg) (10 µL)

R.S.D. 48% ~ 86%

(20 mg), 2-methyl-6-nitro benzoic anhydride (20 mg) 4-dimethylaminopyridine (10 mg)

10 µL 20 µL 6β-OHF 10 µL 20 µL 2.3 10 µL 33% ~ 44% R.S.D. 7% 10, 45, 120 6β-OHF 0.82 ng 10 100% 45 120 101%, 90% 10 1.3.1.2 6β-hydroxycortisol Yamashita 46) _cortisol ₂₁ ₁ 21-mono-picolinyl cortisol

cortisol LC-MS/MS Scan mode

cortisol (MW 362) 1

467 m/z 468

21-mono-picolinyl cortisol

6β-OHF , Yamashita 48)

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13

4 mono-picolinyl 6β-OHF, di-picolinyl 6β-OHF,

tri-picolinyl 6β-OHF, tetra-picolinyl 6β-OHF 6β-OHF 1 ~ 4

[M + H]+

MRM 6β-OHF mono-picolinyl

6β-OHF m/z 484 97%

(Fig. 1-1) di-picolinyl 6β-OHF

m/z 589 2.6% , tri-picolinyl 6β-OHF m/z 694 tetra-picolinyl 6β-OHF m/z 799 mono-picolinyl 6β-OHF 1_H-NMR 6β-OHF 49) 6β-OHF 1 mg TLC TLC

: = 9 : 1 6β-OHF mono-picolinyl 6β-OHF Rf

0.26, 0.40 TLC 3

mL 4 CD3OD 0.5 mL NMR

6β-OHF 6, 11, 17, 21 6, 11, 21 6β-OHF

mono-picolinyl 6β-OHF 1_H-NMR _6α

4.25 ppm (6β-OHF), 4.32 ppm (mono-picolinyl 6β-OHF) 11

mono-picolinyl 6β-OHF: 97.4% di-picolinyl 6β-OHF: 2.6% 0 5 10 15 20 25 m/z 484 m/z 589 m/z 694 m/z 799 (min)

MRM chromatograms of mono-picolinyl 6β-OHF (m/z 484), di-picolinyl 6β-OHF (m/z 589), tri-picolinyl 6β-OHF (m/z 694) and tetra-picolinyl 6β-OHF (m/z 799) in positive ion mode

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14

4.39 ppm (6β-OHF), 4.49 ppm (mono-picolinyl 6β-OHF)

(Fig. 1-2) 21 6β-OHF 4.26 ppm 4.63 ppm mono-picolinyl 6β-OHF 5.27 ppm 5.44 ppm 0.81 ~ 1.01 ppm 6β-OHF mono-picolinyl 6β-OHF 21 mono-picolinyl 6β-OHF 1_H-NMR _{8.09 ppm (3’ ), 8.26} ppm (4’ ), 7.73 ppm (5’ ), 8.75 ppm (6’ ) 4 (A) (B) 1.3.1.3

Fig. 1-3 6β-OHF cortisol,

6β-OHF-2_H

4 cortisol-13C4 MS 6β-OHF, cortisol

Precursor ion [M + H]+ _6β-OHF _{m/z 484, cortisol} _m/z

0 1 2 3 4 5 6 7 8 9 (ppm) 0 1 2 3 4 5 6 7 8 9 (ppm) O OH HO OH OH O 6β-OHF 21 11 6 21-CH2 6α-CH 11α-CH O OH HO OH O O N O mono-picolinyl 6β-OHF 21 11 6 3’ 4’ 5’ 6’ _21-CH₂ 11α-CH 6α-CH

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15 468 Product ion m/z 78, 106, 124

m/z 78 6β-OHF-2_H

4

4 mass units m/z 488 Precursor ion

Product ion m/z 78 cortisol-13_C

4

4 mass units m/z 472 Precursor ion Product ion

m/z 78

1.3.1.4 HPLC

Synergi 4u MAX-RP 80 analytical column (50 × 2.0 mm I.D., 4.0 µm)

Precursor ion 0 50 100 0 100 200 300 400 500 468 (m/z) Int ens it y (%) cortisol 0 50 100 0 100 200 300 400 500 Product ion 78 (m/z) Int ens it y (%) cortisol-13_C 4 0 50 100 0 100 200 300 400 500 472 Precursor ion (m/z) Int ens it y (%) 0 50 100 0 100 200 300 400 500 78 Product ion (m/z) Int ens it y (%) Precursor ion (m/z) 0 50 100 0 100 200 300 400 500 484 Int ens it y (%) 6β-OHF 0 50 100 0 100 200 300 400 500 78 (m/z) Int ens it y (%) Product ion 0 50 100 0 100 200 300 400 500 488 (m/z) Int ens it y (%) Precursor ion 6β-OHF-2_H 4 Product ion 0 50 100 0 100 200 300 400 500 78 (m/z) Int ens it y (%)

Fig. 1-3 Positive-ESI-mass spectra and the product ion spectra of the picolinyl derivatives of

6β-OHF, cortisol, 6β-OHF-2_H

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16

20% (0–2 min), 90% (2.1–6 min), 20% (6.2–15 min) 6β-OHF 0.549 ng/mL cortisol 170.01 ng/mL 6β-OHF 6β-OHF-2_H

4 (7.8 min)

50 mm 250 mm

20% (0–4 min), 65% (4.1–9 min), 90% (9.1–13 min), 20%

(13.2–25 min) 6β-OHF

6β-OHF-2_H 4 7.1–9 min

[20% (0–4 min), 65% (4.1–7 min), 50% (7.1–9 min), 90% (9.1–10 min), 75% (10.2–13

min), 20% (13.2–25 min)] 6β-OHF 6β-OHF-2_H

4 16.64 min, 16.60 min 6β-OHF, 6β-OHF-2_H

4

(Fig. 1-4). cortisol

cortisol-13_C

4 21.01 min 21.00 min

1.3.1.5

Agilent 1200 system (Agilent, Santa Clara. CA, USA) Agilent 6410 triple quadrupole mass spectrometer ESI (+)

6β-OHF 50 pg (Signal to noise ratio: S/N = 3.7) cortisol 2.54 pg (S/N = 4.2)

6β-OHF 1 ng/mL 6β-OHF Time (min) 6β-OHF Int ens it y ( % ) 100 50 0 14 16 18 20 22 24 6β-OHF-2_H 4 Int ens it y ( % ) 100 50 0 Time (min) cortisol Int ens it y ( % ) 100 50 0 Time (min) cortisol-13_C 4 Int ens it y ( % ) 100 50 0 Time (min) (m/z 484→378) (m/z 488→78) (m/z 468→78) (m/z 472→78) ↓ ↓ ↓ ↓ 14 16 18 20 22 24 14 16 18 20 22 24 14 16 18 20 22 24

MRM chromatograms of the picolinyl derivatives of 6β-OHF, cortisol, 6β-OHF-2_H

4and cortisol-13_C

4from plasma samples spiked with 1.49 ng of 6β-OHF-2H4and 54.72 ng of cortisol-13C4in the positive ion mode

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17

6β-OHF S/N 3.7 0.25 pg, cortisol S/N 5.2

0.025 pg LC-MS/MS 6β-OHF

200 cortisol 102 (Fig. 1-5)

6β-OHF (38.51 pg/mL) cortisol (38.10 pg/mL)

(R.E.) (R.S.D.) 6β-OHF, cortisol R.E.

4% R.S.D. 3% 6β-OHF cortisol

(Table 1-1)

Fig. 1-5 Lower limits of detections of picolinyl derivatives of 6β-OHF and cortisol 1 pg (S/N = 9.6) 0.5 pg (S/N = 5.2) 0.25 pg (S/N = 3.7) 0.5 pg (S/N = 11.8) 0.05 pg (S/N = 6.4) 0.025 pg (S/N = 5.2) 6β-OHF cortisol Expected (pg/mL) Found (pg/mL) _R.E. _R.S.D.

Individual values Mean S.D. (%) (%)

6β-OHF 38.51 37.29 37.71 39.55 37.00 36.72 37.74 37.67 1.01 -2.19 2.67

cortisol 38.10 37.53 37.65 36.52 36.51 35.98 36.71 36.82 0.65 -3.37 1.76

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18 1.3.1.6 5 6β-OHF (0.411, 0.822, 1.232, 1.643, 2.465 ng) cortisol (20.22, 60.66, 101.10, 181.98, 252.75 ng/mL) 4 2 100% 7 14 6β-OHF 7 81% ~ 105% 14 90% ~ 120% cortisol 7 78% ~ 113% 14 88% ~ 113% 6β-OHF cortisol 2 (Fig. 1-6)

Fig. 1-6 Stability of picolinyl derivatives of 6β-OHF and cortisol stored at 4°C for 14 days 1.3.1.7 6β-OHF 0.082 ~ 2.465 ng, cortisol 20.22 ~ 252.75 ng 6β-OHF-2_H 4 1.490 ng cortisol-13C4 54.72 ng 6 , x 6β-OHF-2_H 4 6β-OHF cortisol-13_C 4 cortisol y (6β-OHF/6β-OHF-2H4 cortisol/cortisol-13_C 4) 5 1 6β-OHF 0.9995 ± 0.0002 cortisol 0.9997 ± 0.0003 (Table 1-2) 0 20 40 60 80 100 120 140 0 7 14 20.22 ng 60.66 ng 101.10 ng 181.98 ng 252.75 ng 0 20 40 60 80 100 120 140 0 7 14 0.411 ng 0.822 ng 1.232 ng 1.643 ng 2.465 ng (Day) (%) 6β-OHF (Day) (%) cortisol

Compound Range Correlation coefficient Slope Intercept

(ng/mL)

6β-OHF 0.082 - 2.465 0.9995 0.0002 1.0058 0.0661 -0.0038 0.0381

cortisol 20.22 - 252.75 0.9997 0.0003 1.0461 0.0333 -0.0198 0.0491

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19 1.3.1.8 Table 1-3 6β-OHF 0.747 ± 0.002 ng/mL 6β-OHF 0.822 ng 1.484 ± 0.039 ng/mL R.E. 5.41% R.S.D. 2.63% 1.232 ng R.E. 4.14% R.S.D. 3.76% 1 ng 6β-OHF cortisol 63.65 ± 2.19 ng/mL R.E. 1.68%, 3.41% R.S.D. 2.20% 2.09% 6β-OHF cortisol 0.500 ± 0.015 ng/mL 80.17 ± 2.47 ng/mL R.E. 3.3% ~ 1.7% R.S.D. 2.2% ~ 3.1% 6β-OHF cortisol 1.3.2 6β-hydroxycortisol cortisol ( ) 1.3.2.1 6β-hydroxycortisol cortisol LC-MS/MS LC-MS/MS 39)

Added Expected Found (ng/mL) R.E. C.V.

(ng/mL) (ng/mL) Individual values Mean S.D. (%) (%) Intra-day (n = 6) 6β-OHF − − 0.754 0.736 0.749 0.738 0.731 0.773 0.747 0.002 − 2.06 0.822 1.569 1.485 1.427 1.447 1.517 1.515 1.515 1.484 0.039 -5.41 2.63 1.232 1.979 1.943 1.942 1.754 1.908 1.914 1.919 1.897 0.071 -4.14 3.76 cortisol − − 66.38 59.69 63.89 64.57 63.70 63.67 63.65 2.19 − 3.44 40.44 104.09 106.66 103.63 102.43 107.26 108.60 106.47 105.84 2.34 1.68 2.20 80.88 144.53 146.95 149.58 145.30 154.10 151.40 149.44 149.46 3.13 3.41 2.09 Inter-day (n = 5) 6β-OHF − − 0.523 0.508 0.490 0.489 0.489 0.500 0.015 − 3.06 0.822 1.322 1.316 1.340 1.254 1.330 1.340 1.316 0.036 -1.79 2.74 1.232 1.732 1.754 1.683 1.752 1.698 1.675 1.712 0.038 1.72 2.22 cortisol − − 75.45 82.60 81.41 81.08 80.31 80.17 2.47 − 3.08 40.44 120.61 113.47 121.53 122.49 118.23 117.23 118.59 3.61 -1.67 3.04 80.88 161.05 155.33 161.74 153.24 153.29 154.73 155.67 3.51 -3.34 2.26 Table 1-3 Intra-day and inter-day accuracy (R.E.) and precision (R.S.D.) determination of

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20 CYP3A LC MS/MS 6β-OHF cortisol 1.3.1.5 6β-OHF LC-MS/MS ESI (+) 50 pg (S/N = 3.7) 1 ng/mL 6β-OHF 6β-OHF LC-MS/MS

Lutz 50) _{ESI negative ion mode (ESI ( ))}

6β-OHF 1 pg, 0.5 ng/mL

ESI ( ) 6β-OHF

MS Precursor ion

6β-OHF 378 (HCOO ) 45 mass units

m/z 423 [M + HCOO]

Product ion 20 21

m/z 347 ([M − CH2O] ) (Fig. 1-7) 6β-OHF-2_H

4 Precursor ion m/z 427, Product ion [M −

CH2O] m/z 351 6β-OHF 4 mass units

cortisol Precursor ion [M − CH2O]

Product ion cortisol m/z 407, m/z 331, cortisol-13_C

4 m/z 411,

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21 1.3.2.2 HPLC

6β-OHF cortisol

LC-MS/MS cortisol cortisone

Synergi 4u MAX-RP 80 analytical column (75 × 2.0 mm I.D., 4.0 µm)

0.2 mL/min, 0.2% 0.01 M

20% (0 min), 90% (9–10 min), 20% (10.5–15 min)

6β-OHF cortisol 3 6β-OHF (0.411, 1.232, 2.465 ng)

cortisol (20.22, 101.1, 181.98 ng) 75 µL 2 µL

Shimadzu LC/MS 8030 triple quadrupole mass spectrometer

0.411 ng 6β-OHF 6β-OHF 0 50 100 0 100 200 300 400 500 0 50 100 0 100 200 300 400 500 Precursor ion 407 (m/z) Int ens it y ( % ) cortisol Product ion 331 (m/z) Int ens it y ( % ) 0 50 100 0 100 200 300 400 500 0 50 100 0 100 200 300 400 500 cortisol-13_C 4 411 Precursor ion (m/z) Int ens it y ( % ) 335 Product ion (m/z) Int ens it y ( % ) 0 50 100 0 100 200 300 400 500 0 50 100 0 100 200 300 400 500 Precursor ion (m/z) 423 Int ens it y ( % ) 6β-OHF 347 (m/z) Int ens it y ( % ) Product ion 0 50 100 0 100 200 300 400 500 0 50 100 0 100 200 300 400 500 427 (m/z) Int ens it y ( % ) Precursor ion 6β-OHF-2_H 4 Product ion 351 (m/z) Int ens it y ( % )

Fig. 1-7 Negative-ESI-mass spectra and the product ion spectra of 6β-OHF (C.E. 19.0 V), cortisol (C.E. 18.0 eV), 6β-OHF-2_H

(27)

22 6β-OHF 74% ~ 85% 0.2% 0.01 M 0.4% 0.2% 0.01 M 6β-OHF 1.4 ~ 1.6 , cortisol 1.2 ~ 1.5 0.4% 6β-OHF 0.039 ng S/N 6.7 3 R.S.D. 22% 6β-OHF 1 ng/mL 75 µL 2 µL 2 pg 6β-OHF LC-MS/MS 6β-OHF 0.039 ng 75 µL 5 µL 2 µL 1.9 ~ 2.4 3 R.S.D. 9% 10 µL R.S.D. 3% 1.2 20 µL 5 µL 1.9

Synergi 4u MAX-RP 80 analytical column 2.0 mm

5 µL 3.0 mm Kinetex 2.6u

C8 100 column Kinetex 2.6u C8 100 column

1.9 µm 0.35 µm

Synergi 4u

MAX-RP 80 analytical column 1.0 mm 0.2 mL/min

0.0637 mm/min 1.5 mm Kinetex 2.6u C8 100

column 0.0637 mm/min 0.45 mL/min

0.2 mL/min 0.45 mL/min 6β-OHF

0.039 ng 75 µL 20 µL R.S.D. 1%

(Fig. 1-8) 6β-OHF cortisol 1.49 ng/mL, 54.72

(28)

23 1.3.2.3 6β-OHF S/N = 9.18 5.15 pg S/N = 7.51 2.16 pg 0.25 pg (S/N = 3.7) 10 1.3.2.2 75 µL 20 µL 8.1 pg/mL 6β-OHF cortisol S/N = 5.62 2.04 pg cortisol (Fig. 1-9) Time (min) 6β-OHF Int ens it y ( % ) 100 50 0 1 2 3 4 5 6 7 1 2 3 4 5 6 7 6β-OHF-2_H 4 Int ens it y ( % ) 100 50 0 Time (min) 1 2 3 4 5 6 7 cortisol Int ens it y ( % ) 100 50 0 Time (min) 1 2 3 4 5 6 7 cortisol-13_C 4 Int ens it y ( % ) 100 50 0 Time (min) (m/z 423→347) (m/z 427→351) (m/z 407→331) (m/z 411→335) ↓ ↓ ↓ ↓ 6β-OHF cortisol 5.15 pg (S/N = 9.18) (S/N = 7.51)2.16 pg (S/N = 9.01)5.10 pg (S/N = 5.62)2.04 pg 393 3.0 3.5 423.20>347.20(-)@ 2 1,129 6.5 407.00>331.25(-)@ 1 ₃₁₄ 3.0 3.5 423.20>347.20(-)@ 2 868 6.5 407.00>331.25(-)@ 1 178 3.0 3.5 423.20>347.20(-)@ 2 505 6.5 407.00>331.25(-)@ 1 99 3.0 3.5 423.20>347.20(-)@ 2 372 6.5 407.00>331.25(-)@ 1

Fig. 1-9 Lower limits of detections of picolinyl nonderivatives of 6β-OHF and cortisol

MRM chromatograms of 6β-OHF, cortisol, 6β-OHF-2_H

4and cortisol-13C4from plasma

samples spiked with 1.49 ng of 6β-OHF-2_H

4 and 54.72 ng of cortisol-13C4 in the

(29)

24

Expected (pg/mL)

Found (pg/mL) _R.E. _R.S.D.

6β-OHF 38.51 33.91 38.19 38.97 37.73 37.09 36.12 37.00 1.80 -3.92 4.86

cortisol 38.10 37.50 37.39 36.89 37.74 36.97 37.26 37.29 0.32 -2.12 0.86

Table 1-4 Lower limits of quantification of the LC-MS/MS analyses of 6β-OHF (38.51 pg/mL) and cortisol (38.10 pg/mL) using nonderivatization method (n = 6)

38.51 pg/mL (6β-OHF), 38.10 pg/mL (cortisol) R.E. 4% R.S.D. 5% (Table 1-4) Barret 38) _6β-OHF _LC-MS/MS _{ESI ( )}

1.0 ng/mL Barret 26 1 38.51 pg/mL 6β-OHF cortisol Barret (0.2 ng/mL) 5 1 (38.10 pg/mL) 1.3.2.4 3 LC-MS/MS 6β-OHF 0.742 ng/mL, 1.064 ng/mL, 1.523 ng/mL cortisol 101.94 ng/mL, 138.70 ng/mL, 179.22 ng/mL 20 21 48 100% 21 91% ~ 102% (6β-OHF) 100% ~ 103%

(cortisol) 48 95% ~ 101% (6β-OHF) 96% ~ 100% (cortisol)

1.5 (Fig. 1-10) 0 20 40 60 80 100 120 140 0 21 42 101.94 ng 138.70 ng 179.22 ng 0 20 40 60 80 100 120 140 0 21 42 0.7418 ng 1.0636 ng 1.5233 ng (Day) (%) 6β-OHF cortisol (Day) (%)

(30)

25 4 100% 3, 5, 7 6β-OHF 90% ~ 120%, cortisol 88% ~ 113% 4 1.3.2.5 6β-OHF 0.082 ~ 2.465 ng, cortisol 20.22 ~ 252.75 ng 6β-OHF-2_H 4 1.490 ng cortisol-13C4 54.72 ng 6 , x 6β-OHF-2_H 4 6β-OHF cortisol-13_C 4 cortisol y (6β-OHF/6β-OHF-2H4 cortisol/cortisol-13_C 4) 5 1 6β-OHF 0.9997 ± 0.0002 cortisol 0.9997 ± 0.0002 (Table 1-5) 1.3.2.6 Table 1-6 6β-OHF cortisol 0.420 ± 0.016 ng/mL, 38.36 ± 0.12 ng/mL R.E. 4.9% ~ 0.2% R.S.D. 0.3% ~ 3.9% 6β-OHF cortisol 0.409 ± 0.011 ng/mL 38.38 ± 0.34 ng/mL R.E. 1.9% ~ 0.7% R.S.D. 0.9% ~ 2.8% 1.3.1.8 6β-OHF, cortisol

Compound Range Correlation coefficient Slope Intercept

(ng/mL)

6β-OHF 0.082 - 2.465 0.9997 0.0002 1.0096 0.0254 -0.0035 0.0081

cortisol 20.22 - 252.75 0.9997 0.0002 1.0122 0.0214 -0.0132 0.0408

(31)

26 1.3.3 6β-hydroxycortisol cortisol 1.3.3.1 LC-MS/MS 51,52)_. LC-MS/MS (6β-OHF-2_H 4 cortisol-13C4) 53) 13_C 2_H 2_H 1_H ₂ 13_C 12_C 13_C 2_H _cortisol _cortisol-13_C 4 21.02 min 21.00 min

5.99 min 6β-OHF 6β-OHF-2_H

4

16.64 min, 16.60 min 2.68 min,

2.65 min cortisol cortisol-13_C

4 6β-OHF

Added Expected Found (ng/mL) R.E. C.V.

(ng/mL) (ng/mL) Individual values Mean S.D. (%) (%) Intra-day (n = 6) 6β-OHF − − 0.397 0.421 0.430 0.419 0.407 0.444 0.420 0.016 − 3.92 0.822 1.242 1.153 1.236 1.198 1.158 1.148 1.205 1.183 0.035 -4.73 2.99 1.232 1.652 1.591 1.585 1.546 1.609 1.556 1.538 1.571 0.028 -4.90 1.80 cortisol − 38.48 38.34 38.50 38.41 38.19 38.25 38.36 0.12 0.32 40.44 78.80 78.84 77.68 78.11 79.60 77.44 77.84 78.25 0.82 -0.70 1.04 80.88 119.24 120.10 119.01 118.92 119.34 120.00 119.44 119.47 0.49 0.19 0.41 Inter-day (n = 5) 6β-OHF − 0.420 0.414 0.393 0.403 0.418 0.409 0.011 − 2.77 0.822 1.231 1.183 1.220 1.192 1.230 1.220 1.209 0.020 -1.85 1.67 1.232 1.641 1.571 1.640 1.637 1.611 1.695 1.631 0.045 -0.65 2.78 cortisol − 38.36 37.87 38.33 38.55 38.79 38.38 0.34 − 0.88 40.44 78.82 78.25 77.83 75.71 78.21 76.94 77.39 1.08 -1.82 1.39 80.88 119.26 119.47 118.91 116.17 118.87 116.97 118.08 1.43 -0.99 1.21 Table 1-6 Intra-day and inter-day accuracy (R.E.) and precision (R.S.D.) determination of

(32)

27 1.3.3.2 6β-OHF cortisol 6β-OHF cortisol (n = 75) 6β-OHF 0.678 ± 0.326 ng/mL (0.173 ~ 1.549 ng/mL), 0.723 ± 0.326 ng/mL (0.227 ~ 1.653 ng/mL) 2 ng/mL cortisol 117.16 ± 66.15 ng/mL (23.78 ~ 294.71 ng/mL), 117.21 ± 63.54 ng/mL (30.95 ~ 308.54 ng/mL) LC-MS/MS 1.3.1.8 1.3.2.6 6β-OHF cortisol x y Fig. 1-11 y = 0.9618x - 0.017 (6β-OHF), y = 1.0268x - 3.1926 (cortisol), r = 0.9613 (6β-OHF), r = 0.9862 (cortisol) 6β-OHF, cortisol 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 plasma cortisol (ng/mL) by nonderivatization method plasma 6β-OHF (ng/mL) by nonderivatization method pla sma 6 β-O H F ( ng/ m L ) by de ri va ti za ti on m et hod pla sma 6 β-O H F ( ng/ m L ) by de ri va ti za ti on m et hod

(A)

(B)

y r = 0.9618x – 0.017= 0.9613 y r = 1.0268x – 3.1926= 0.9862

Correlations between the picolynil derivatization and nonderivatization methods. Linear regression data for the plasma concentrations measured by LC-MS/MS for nonderivatized (x-axis; ng/mL) and picolinyl derivatized (y-axis; ng/mL) 6β-OHF (A) and cortisol (B).

(33)

28

CYP3A 6β-OHF

6β-OHF/cortisol CYP3A

(34)

29

2 6β-hydroxycortisol /cortisol CYP3A

2.1 P450 3A (CYP3A) 5 ~ 20 CYP3A 2,3) CYP3A 1 CYP3A

CYP3A Gold standard

CYP3A 12) 1 cholesterol CYP3A 4β-hydroxycholesterol 4β-hydroxycholesterol 4β-hydroxycholesterol/cholesterol 17) CYP3A 18) CYP3A 19) 4β-hydroxycholesterol 17 20) _cortisol

CYP3A 6β-hydroxycortisol (6β-OHF) 6β-OHF/cortisol

23) 24) 26) cortisol 6β-OHF (CLm(6β)) CYP3A 26) _CL m(6β) 6β-OHF cortisol (AUC) cortisol

cortisol CYP3A cortisol

(35)

30

CYP3A CLm(6β)

CYP3A

CYP3A 6β-OHF cortisol

1 CYP3A CYP3A CLm(6β) 1 6β-OHF/cortisol in vivo CYP3A 1 6β-OHF/cortisol CYP3A 2.2 2.2.1 6β-hydroxycortisol/cortisol CYP3A 2.2.1.1 54)

21®_{(ASKA pharmaceutical Co., Ltd. Tokyo, Japan)} 3 (Subject A: 33 , Subject B: 28 , Subject C: 31 )

(36)

31 Subject C 2 (Day 2) 8 18 1 2 ( : 17-1, : 17 9 21 29 4 1 ) ( : 2078, : 22 12 14 ) 2.2.1.2 6β-hydroxycortisol cortisol

6β-hydroxycortisol (6β-OHF) cortisol 1 LC-MS/MS

2.2.1.3 cortisol 6β- (CLm(6β)) 54)

CLm(6β) CLm(6β) 2

6β-OHF cortisol

-(AUC) cortisol GC-MS 6β-OHF HPLC-UV

(37)

32 2.2.3 6β-hydroxycortisol/cortisol CYP3A 20 20 2 CYP3A CYP3A 1 (BSA) 70 mg/m2 _{(0 hr),} 1 6 9 24 2.3 2.3.1 6β-hydroxycortisol/cortisol CYP3A

CYP3A in vivo Gold standard

CYP3A 1’- p-CYP3A Hohmann 13) 1 ~ 3 mg 1000 1 1 ~ 3 µg 1’-CYP3A Lin 14) _(AUC) ₄ 4 CYP3A 1 CYP3A

(38)

33

cholesterol CYP3A 4β-hydroxycholesterol 2008

Diczfalusy 17) _CYP3A 4β-hydroxycholesterol 4β-hydroxycholesterol/cholesterol CYP3A 21,22) CYP3A 4β-hydroxycholesterol CYP3A 27) 4β-hydroxycholesterol 17 1 20)

cortisol CYP3A 6β-OHF

cortisol 6β-OHF CYP3A

6β-OHF/cortisol cortisol 26)

CYP3A

1 CYP3A 1

6β-OHF/cortisol Shin 27) _CYP3A

cortisol, teststerone, estradiol

(39)

34

CYP3A 3

CYP3A

2.3.1.1 6β-hydroxycortisol cortisol

Fig. 2-1 3 6β-OHF

cortisol cortisol Subject A Day 0 51.82

~ 121.33 ng/mL Day 1 44.41 ~ 129.04 ng/mL 21

(Day 21) 13 Day 0, Day 1 8 ~ 12

148.37 ~ 305.11 ng/mL Day 0, Day 1

7 Day 28 Day0, Day 1 54.66 ~ 142.73 ng/mL

Subject B, Subject C Day 21 Subject

B 4 33.54 ~ 271.36 ng/mL Subject C Day 2 5

23.78 ~ 294.71 ng/mL ethinylestradiol

cortisol 55) _{Day 21}

cortisol

ethinylestradiol

6β-OHF Subject A Day 0, Day 1, Day 28 0.283 ~ 1.017 ng/mL,

Day 21 3 0.328 ~ 1.781 ng/mL

cortisol Subject B 0.447 ~ 1.549 ng/mL, Subject

C 0.275 ~ 0.938 ng/mL CYP3A cortisol

6β-OHF cortisol 1% 29) ₃ _{Day 0}

6β-OHF cortisol 0.5 ~ 1.0% (Subject A) 0.4 ~ 1.4% (Subject B) 0.4 ~

0.9% (Subject C) 6β-OHF

1.4 ~ 5.4 6β-OHF Varga 56)

36 8 ~ 9 6β-OHF

0.62 ± 0.04 ng/mL Imamura 37)

(40)

35 Subject A

Subject B

Subject C

(41)

36 2.3.1.2 6β-hydroxycortisol/cortisol

Fig. 2-2 3 6β-OHF/cortisol

CLm(6β) Day 0 Day 1 6β-OHF/cortisol

Subject A 0.0077 ± 0.0013 (0.0052 ~ 0.0096: Day 0), 0.0097 ± 0.0019 (0.0077 ~ 0.0134: Day 1), Subject B 0.0111 ± 0.0032 (0.0039 ~ 0.0136: Day 0), 0.0106 ± 0.0044 (0.0028 ~ 0.0148: Day 1), Subject C 0.0066 ± 0.0016 (0.0044 ~ 0.0088: Day 0), 0.0064 ± 0.0013 (0.0042 ~ 0.0087: Day 1) 3 Day 0 Day 1

6β-OHF/cortisol 1 CYP3A

Subject C Day 2 0.0054 ± 0.0013 (0.0036 ~ 0.0082)

Day 0, Day 1 6β-OHF/cortisol

3 Day 0 1 1.7

21 (Day 21) 6β-OHF/cortisol Subject A 0.0047 ± 0.0012 (0.0032 ~ 0.0065), Subject B 0.0057 ± 0.0006 (0.0049 ~ 0.0065), Subject C 0.0026 ± 0.0003 (0.0019 ~ 0.0029) CYP3A Day 0 61%, 51%, 39% 7 Day 28 Subject A 0.0051 ± 0.0010 (0.0030 ~ 0.0061), Subject B 0.0084 ± 0.0016 (0.0054 ~ 0.0110), Subject C 0.0035 ± 0.0006 (0.0021 ~ 0.0042) Day 21 Day 0 66%, 76%, 53% CLm(6β) CYP3A Day 21

CLm(6β) Day 0 57%, 46%, 41% Day 28 Day 0 68%, 76%,

63% 3 6β-OHF/cortisol CLm(6β) 6β-OHF/cortisol CYP3A 1 2 CLm(6β) 2 6β-OHF cortisol (AUC) AUC ( ), 1 2 ( ) 3 cortisol CLm(6β) 3 2 54) _{6β-OHF/cortisol} 10:00 ~ 18:00 6β-OHF/cortisol 9 CLm(6β) 4 6β-OHF/cortisol CLm(6β) CYP3A

3 6β-OHF/cortisol Subject A Subject C 1.3 ~

2.3 CLm(6β)

(1.5 ~ 3.0 ) Subject B Day 0 3.4 Day 1 5.2

Fig. 2-2 6β-OHF cortisol

(42)

37 cortisol cortisol cortisol 1 6β-OHF/cortisol cortisol

Subject B Day 0, Day 1 13 cortisol

(43)

38 Subject A

Subject B

Subject C

Time course of plasma 6β-OHF/cortisol ratio and CLm(6β)in three healthy subjects before, during and after taking oral contraceptives

(44)

39

2.3.1.3 CLm(6β) 6β-hydroxycortisol/cortisol Watkins 7)

cortisol 6β-OHF CYP3A

CLm(6β) 6β-OHF/cortisol in vivo CYP3A x CLm(6β) y 6β-OHF/cortisol 6β-OHF/cortisol 2 3 Subject A Subject B 18 Subject C 23 59 6β-OHF/cortisol CLm(6β) (r = 0.9054) (Fig. 2-3)

6β-OHF/cortisol in vivo CYP3A

6β-OHF/cortisol CLm(6β) 0.0035, 0.0039, 0.0045 CLm(6β) x 6β-hydroxycortisol/cortisol y

CL

m(6β)

(mL/min)

pl

am

sa

6β

-OHF

/c

or

ti

sol

rat

io

y = 0.0039x + 0.0011

r = 0.9054

0.000 0.002 0.004 0.006 0.008 0.010 0.012 0.014 0.016 0.018 0.020 0.0 1.0 2.0 3.0 4.0

(45)

40

CLm(6β) Limited sampling strategy cortisol

(AUC) cortisol AUC

2 cortisol AUC (AUCcortisol) 30 5

AUCcortisol 2 1

(2 ) (Prediction error: P.E.)

14.3% AUCcortisol AUCcortisol 2

1 CLm(6β)

6β-OHF/cortisol 2 1

(1)

C6β-OHF × T2hr 6β-OHF AUC

C6β-OHF; 6β-OHF Ccortisol; cortisol T2hr; (2 ) X6β-OHF; 6β-OHF

Slope = 6β-OHF/cortisol ratio in plasma

CL_m(6β) (1) C_6β-OHF C_cortisol × AUC_cortisol X_6β-OHF = C6β-OHF C_cortisol × C_cortisol× T_2hr X_6β-OHF = C6β-OHF × T2hr X_6β-OHF Slope =

(46)

41

6β-OHF (CLR(6β-OHF))

CLm(6β) 6β-OHF 6β-OHF/cortisol

6β-OHF Subject A, B, C 1 CL

R(6β-OHF) CLR(6β-OHF) 8 10 6β-OHF

6β-OHF (AUC6β-OHF) Subject A, Subject

B Subject C CLR(6β-OHF) 229.01 ± 12.79 mL/min, 243.46 ± 49.74 mL/min, 199.25 ± 23.23 mL/min R.S.D. 5.6% ~ 20.4%,

R.S.D. 10.1% (Table 2-1) Imamura 37) _6β-OHF OAT3 6β-OHF OAT3 6β-OHF 231 ± 11 mL/min 3 6β-OHF OAT3 OAT 3 6β-OHF 6β-OHF 6β-OHF/cortisol 6β-OHF 6β-OHF/cortisol OAT3 3 CLm(6β) 6β-OHF/cortisol CYP3A 6β-OHF/cortisol CYP3A CLm(6β) 6β-OHF/cortisol CLm(6β) (r = 0.9054) 6β-OHF/cortisol CYP3A

Table 2-1 CLR(6β-OHF)(mL/min) in three healthy subjects

Day 0 Day 1 Day 2 Day21 Day 28 Mean S.D. R.S.D. (%)

Subject A 220.64 230.46 215.77 249.18 229.01 12.79 5.58

Subject B 281.61 302.74 185.28 204.22 243.46 49.74 20.43

(47)

42 2.3.2 6β-hydroxycortisol/cortisol 2.3.2.1 6β-hydroxycortisol/cortisol 2 (Subject D, E) 28 1 6β-OHF/cortisol CYP3A 10, 11, 12, 16, 17, 18

Fig. 2-4 2 1 6β-OHF cortisol

. cortisol Subject D 26.68 ~ 214.88 ng/mL Subject E 27.33 ~

155.76 ng/mL cortisol Subject D 1.6 ~ 4.3 Subject E 1.6 ~ 3.5 cortisol 57)_. ₃ 30 24 5 ~ 180 ng/mL 58) ₆ ₁₁ 6 11 3 1 6β-OHF 1 6 1 6β-OHF Subject D 0.259 ~ 1.473 ng/mL, Subject E 0.360 ~ 1.117 ng/mL

cortisol 6β-OHF Subject D 1.9 ±

0.5 (1.1 ~ 3.3 ) Subject E 1.7 ± 0.2 (1.6 ~ 2.2 ) 1

6β-OHF Subject D 1.3 Subject E 1.4

6β-OHF/cortisol 1 Subject D 0.0047 ~ 0.0132 Subject

(48)

43

Diurnal and day-to-day variations of plasma 6β-OHF and cortisol concentrations in two subjects between Day 1 ~ 364

Fig. 2-4

Diurnal and day-to-day variations of plasma 6β-OHF/cortisol ratio in Subject D and Subject E between Day 1 ~ 364

(49)

44 2.3.2.2 6β-hydroxycortisol/cortisol 22 ( 10 , 12 : 22 ~ 55 ) CYP3A 6β-OHF/cortisol 22 2 Subject D, E 2.3.2.1 6β-OHF/cortisol 20 10:00 ~ 12:00 1 6β-OHF/cortisol 22 6β-OHF/cortisol 0.0093 ± 0.0030 (Mean ± S.D.) 0.0059 ~ 0.0172 (Fig. 2-6) 6β-OHF/cortisol 2.9 21 CLm(6β) (3.5 ) 22 (10 ), (12 ) 0.0079 ± 0.0019 (0.0059 ~ 0.0116) 0.0104 ± 0.0033 (0.0073 ~ 0.0172) (p = 0.0449) CYP3A

(50)

45

(51)

46 6β-OHF 1.021 ± 0.774 ng/mL 20 1 3.794 ng/mL 19 6β-OHF 0.350 ~ 1.855 ng/mL 6β-OHF 6β-OHF/cortisol 19 6β-OHF/cortisol 0.0071 ~ 0.0180 6β-OHF/cortisol CYP3A 6β-OHF/cortisol (Fig. 2-7) CYP3A 18,60,61) 6β-OHF/cortisol CYP3A 20 6β-OHF/cortisol 6β-OHF/cortisol 0.0003 ~ 0.0349 1.21 ~ 47.7 3 3 Fig. 2-8 Patient A 6β-OHF/cortisol 0.0119 ~ 0.0195 20 15 6β-OHF/cortisol Patient B 0.0220 0 10 20 30 40 50 0 0.01 0.02 0.03 (n = 20)

plasma 6β-OHF/cortisol ratio

doc et axe l cl ea ra nc e ( L /hr )

(52)

47 ~ 0.0349 24 20 3 Patient C 1 0.0098, 0.0116 6β-OHF/cortisol 6 0.0003 ~ 0.0006 1 CYP3A 6β-OHF/cortisol 1 CYP3A CYP3A

(53)

48

3 Dried blood spots 6β-hydroxycortisol/cortisol CYP3A

3.1

(Dried blood spots: DBS)

(54)

49 3.2

3.2.1

1.2.1 1.2.2

6β-hydroxycortisol (6β-OHF) cortisol 0.00021 µg/mL, 0.101

µg/mL 6β-OHF-2_H

4 (0.00298 µg/mL), cortisol-13_C

4 (0.182 µg/mL)

3.2.2 LC-MS/MS

LC-MS/MS LC/MS 8030 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan) LC-20AD binary gradient pump, SIP-20A auto-sampler, DGU-20A3 degasser,

CTO-20AC column oven LC system

Phenomenex (CA, USA) Kinetex 2.6u C8 100 column (75 × 3.0 mm I.D., 2.6 µm),

Security Guard ULTRA Cartridge C8 (3.0 mm I.D.) 40

0.45 mL/min 0.0125%

20% (0 min), 90% (9–10 min), 20% (10.5–15 min)

15 Electrospray ionization (ESI) Nebulizer gas (3

L/min) Drying gas (15 L/min) Collision voltage 6 kV, Source temperature 400 , Desolvation temperature 290 Multiple reaction monitoring (MRM) Negative ion mode Precursor ion Product ion

6β-OHF m/z 423 347 6β-OHF-2_H

4 m/z 427 351 cortisol m/z 407

331, cortisol-13_C

4 m/z 411 335 Collision energy 6β-OHF

6β-OHF-2_H

4 19 eV cortisol cortisol-13C4 15 eV 3.2.3 Dried blood spots

49 ,

37243002 (Asahi Polyslider, Okayama, Japan)

1 15 ~ 25 µL

(Dried blood spots: DBS) DBS 1

3.2.4

(55)

50 3 mL 6β-OHF-2_H 4 (0.0894 ng), cortisol-13C4 (5.47 ng) (1500 rpm/30 sec) (1 mL × 3 ) (50 µL) 40 µL LC-MS/MS 3.2.5

(56)

51

vivo CYP3A 6 ( 3 , 3 ) 8 mL (3000 rpm/10 min) DBS 1 6β-OHF cortisol DBS 3.2.4 DBS LC-MS/MS 3.3 3.3.1 1 2 6β-OHF 0.275 ~ 1.781 ng/mL

(Dried blood spots: DBS) DBS 1

15 ~ 25 µL DBS 6β-hydroxycortisol

(6β-OHF) 1

6β-OHF 0.25 pg

38.51 pg/mL DBS 6β-OHF cortisol

6β-OHF ( 16.64 min) cortisol ( 20.91 min) (Fig. 3-1) DBS DBS 6β-OHF 分析日時 : 2018/07/09 16:07:47 サンプル名 : D B S -02 バイアル番号 : 93 2,053 5.0 5.5 6.0 6.5 7.0 484.00>77.95(+)@ 1 200,000 6.0 6.5 7.0 7.5 8.0 468.00>77.90(+)@ 3 分析日時 : 2018/07/09 16:07:47 サンプル名 : D B S-02 バイアル番号 : 93 2,053 5.0 5.5 6.0 6.5 7.0 484.00>77.95(+)@ 1 200,000 6.0 6.5 7.0 7.5 8.0 468.00>77.90(+)@ 3 In te n si ty (%) 0 100 50 14 16 18 20 22 24 Time (min) cortisol In te n si ty (%) 100 50 0 14 16 18 20 22 24 Time (min) (m/z 484→78) (m/z 468→78) 6β-OHF

↓

MRM chromatograms of picolinyl derivatives of 6β-OHF, cortisol from DBS samples in the positive ion mode

(57)

52 DBS 4 1 4 6β-OHF 4 60 ~ 100 µL 30 ~ 50 µL 6β-OHF 10 ~ 50 pg 6β-OHF 2.16 pg, 38.51 pg/mL DBS DBS 6β-OHF cortisol 6β-OHF Electrospray ionization (ESI) 0.4% 0.1%, 0.05%, 0.025%, 0.0125%, 0.0065% 0.4% 100 6β-OHF 0.1% 0.05% 332, 0.025% 497 0.0125% 1048 0.4% 10.5 (Fig. 3-2) 0.0065% 0.0125%

1 Kinetex 2.6u C8 100 column (75 × 3.0 mm I.D.,

2.6 µm) LC-MS/MS

1 ~ 10 µL DBS 6β-OHF

(58)

53 DBS 6β-OHF 0.0125% LC-MS/MS DBS 6β-OHF 5.12 pg 6β-OHF 50 µL 10 µL LC-MS/MS 3 (R.S.D.) 15% 20 µL, 40 µL 40 µL 4 LC-MS/MS R.S.D. 3% (Fig. 3-3) 50 µL 40 µL

Fig. 3-3 MRM chromatograms of 6β-OHF by injecting different volume

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54

DBS 3.2.4 LC-MS/MS

6β-OHF cortisol Fig. 3-4 2.64 min

6β-OHF 5.88 min cortisol

DBS 0.0188 ng (6β-OHF), 2.10 ng (cortisol) 3.3.2 DBS DBS 4 1 30,67) _6β-OHF DBS 1 1 4 DBS DBS Odoardi 69) _DBS Nirogi 70) _HIV DBS 6β-OHF 2,423 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 423.20>347.20(-)@ 1 2,585 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 427.10>351.25(-)@ 2 321,789 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 407.00>331.25(-)@ 3 531,223 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 411.00>335.25(-)@ 4 2,423 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 423.20>347.20(-)@ 1 2,585 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 427.10>351.25(-)@ 2 321,789 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 407.00>331.25(-)@ 3 531,223 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 411.00>335.25(-)@ 4 2,423 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 423.20>347.20(-)@ 1 2,585 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 427.10>351.25(-)@ 2 321,789 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 407.00>331.25(-)@ 3 531,223 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 411.00>335.25(-)@ 4 2,423 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 423.20>347.20(-)@ 1 2,585 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 427.10>351.25(-)@ 2 321,789 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 407.00>331.25(-)@ 3 531,223 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 411.00>335.25(-)@ 4 Time (min) 6β-OHF Int ens it y (%) 100 50 0 2 3 4 5 6 7 8 2 3 4 5 6 7 8 6β-OHF-2_H 4 Int ens it y (%) 100 50 0 Time (min) 2 3 4 5 6 7 8 cortisol Int ens it y (%) 100 50 0 Time (min) 2 3 4 5 6 7 8 cortisol-13_C 4 Int ens it y (%) 100 50 0 Time (min) (m/z 423→347) (m/z 407→331) (m/z 411→335) (m/z 427→351)

MRM chromatograms of 6β-OHF, cortisol, 6β-OHF-2_H

4and cortisol-13C4from DBS spiked with 89.4 pg of 6β-OHF-2_H

4and 5.47 ng of cortisol-13C4in the negative ion mode

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55 (0.0161 ng) cortisol (2.43 ng) 3 mL (1 mL × 3 ) DBS (1 mL × 3 ) 50 µL 40 µL LC-MS/MS 96% ~ 106%, R.S.D. 0.58% ~ 7.82% (Table 3-1) cortisol 6.2%, 6β-OHF 96% ~ 99%, 105% ~ 106% 1 ,

Table 3-1 Extraction rate of 6β-OHF and cortisol using methanol, water and ethyl acetate

3.3.3 6β-hydroxycortisol cortisol DBS 7 D 1 ~ 2 30,71) _6β-OHF _cortisol _DBS 12 4 3.2.4 LC-MS/MS 8 7 28 4 7 28 DBS 6β-OHF 0.0388 ± 0.0011 ng, cortisol 18.60 ± 0.36 ng

Compound Extraction Added Found (ng) Extraction rate R.S.D.

solvent (ng) _{Individual values} _Mean _S.D. (%) (%)

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56 7 98.1% ~ 101.6% 28 99.7% ~ 103.3% DBS 6β-OHF cortisol 28 DBS 30) _DBS DBS 3.3.4 LC-MS/MS DBS DBS 72) _6β-OHF-2_H 4 (0.0894 ng) cortisol-13C4 (5.47 ng) (Sample A) (Sample B) DBS (Subject B) 1 mL 6β-OHF-2_H 4 (0.0894 ng) cortisol-13C4 (5.47 ng) DBS 3.2.4 DBS LC-MS/MS

Sample A 6β-OHF cortisol Sample B

(R.E.) 6β-OHF −2.09%, cortisol −4.42% R.S.D. 5.07% ~ 12.69%

(Table 3-2) 3.2.2 105% ~ 106%

Sample A Sample B R.E. R.S.D.

3.2.5

6β-OHF 0.0051 ~ 0.1230 ng, cortisol 2.02 ~ 12.12 ng 6β-OHF-2_H

4 0.0894 ng cortisol-13C4 5.47 ng Table 3-2 Amount of 6β-OHF and cortisol in Sample A and Sample B

Compound Sample Found (ng) R.E. R.S.D.

6β-OHF 0.0821 0.0655 0.0673 0.0717 0.0091 12.69

B 0.0737 0.0736 0.0632 0.0702 0.0061 -2.09 8.64

cortisol A 5.82 5.27 5.66 5.58 0.28 5.07

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57 6 x 6β-OHF-2_H 4 6β-OHF cortisol-13_C 4 cortisol y (6β-OHF/6β-OHF-2_H 4 cortisol/cortisol-13C4) 5 6β-OHF 0.9982 ± 0.0010 cortisol 0.9997 ± 0.0002 (Table 3-3) 3.2.6 Table 3-4 DBS 6β-OHF 0.0962 ± 0.0016 ng R.S.D. 1.71% DBS 6β-OHF 0.0054 ng 0.1074 ± 0.0057 ng R.E. 5.68% R.S.D. 5.29% 4 DBS 6β-OHF

cortisol cortisol 7.62 ± 0.20 ng cortisol 0.81 ng

R.E. 1.89% R.S.D. 2.73% cortisol

3.3.7 6β-hydroxycortisol cortisol

DBS

DBS DBS

6β-OHF Subject A 0.0455 ng ( ), 0.0440 ng ( ) Subject B 0.0566 ng (

) 00560 ng ( ) DBS 6β-OHF cortisol

Table 3-3 Linearity date for the LC-MS/MS analysis of 6β-OHF and cortisol (n = 5)

Table 3-4 Accuracy (R.E.) and precision (R.S.D.) determination of 6β-OHF and cortisol in DBS

Added Expected Found (ng) R.E. R.S.D.

(ng) (ng) _{Individual values} _Mean _S.D. (%) (%)

6β-OHF − − 0.0950 0.0956 0.0981 0.0962 0.0016 − 1.71

0.0054 0.1016 0.1076 0.1129 0.1016 0.1074 0.0057 5.68 5.29

cortisol − − 7.75 7.40 7.73 7.62 0.20 − 2.59

0.81 8.43 8.86 8.45 8.47 8.59 0.23 1.89 2.73

Compound Range (ng) Correlation_coefficient Slope Intercept

6β-OHF 0.0051 - 0.1230 0.9982 0.0010 0.9972 0.0270 -0.0205 0.0135

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58 Subject A Subject B DBS (ng) Whole blood (ng/mL) Plasma DBS (ng) Whole blood (ng/mL) Plasma Left hand Right hand (ng/mL) Left hand Right hand (ng/mL) 6β-OHF 0.0455 0.0440 0.441 0.577 0.0566 0.0560 0.636 0.810 cortisol 3.37 3.33 30.47 48.88 4.18 4.54 44.20 74.49 6β-OHF/cortisol ratio 0.0135 0.0132 0.0145 0.0118 0.0135 0.0123 0.0144 0.0109 Subject A 3.37 ng ( ), 3.33 ng ( ) Subject B 4.18 ng ( ) 4.54 ng ( ) DBS 30,66) 40% ~ 52% 33% ~ 45% DBS 30,66,73) Table 3-5 2 DBS, 6β-OHF

cortisol Subject A 6β-OHF DBS 0.0455 ng

0.441 ng/mL 0.577 ng/mL 65% cortisol DBS 3.37 ng 30.47 ng/mL 48.88 ng/mL 80% Subject B 6β-OHF DBS 0.0566 pg, 0.636 ng/mL 0.810 ng/mL cortisol DBS 4.18 ng 44.20 ng/mL 74.49 ng/mL 6β-OHF cortisol 65%, 85%

vivo CYP3A

6 ( 3 , 3 ) DBS 6β-OHF/cortisol

Subject A ~ D 2 DBS

Subject E, F 1 DBS Subject A, E, F

Subject B, C, D

Amount of 6β-OHF and cortisol and 6β-OHF/cortisol ratio in two healthy subjects

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59

2 6β-OHF/cortisol CYP3A

DBS

6β-OHF cortisol

Table 3-6 6 (n = 10) DBS 6β-OHF

cortisol 6β-OHF/cortisol DBS 6β-OHF 0.0101 ~ 0.0507

ng DBS 6β-OHF cortisol 1.04 ~ 3.76 ng 6 10 6β-OHF/cortisol 0.0087 ± 0.0022 (0.0060 ~ 0.00123) 2 6β-OHF/cortisol DBS 6β-OHF/cortisol 0.0125 ± 0.0036 (0.0072 ~ 0.00189) 6β-OHF ( 65%) cortisol ( 80%) DBS 6β-OHF/cortisol x 6β-OHF/cortisol y DBS 6β-OHF/cortisol (Fig. 3-5) y = 1.5447x - 0.0012 r = 0.9548 DBS 6β-OHF/cortisol

DBS 6β-OHF/cortisol in vivo CYP3A

DBS Plasma

6β-OHF cortisol 6β-OHF/cortisol ratio

6β-OHF cortisol 6β-OHF/cortisol ratio (ng) (ng) (ng/mL) (ng/mL) Subject A 0.0101 1.04 0.0097 0.2820 34.90 0.0081 0.0349 2.95 0.0118 0.7426 104.54 0.0071 Subject B 0.0283 2.08 0.0136 0.7414 80.38 0.0092 0.0382 2.29 0.0167 0.8046 65.24 0.0123 Subject C 0.0235 3.28 0.0072 0.5314 89.30 0.0060 0.0444 3.76 0.0118 0.9266 109.90 0.0084 Subject D 0.0226 1.72 0.0131 0.7414 80.40 0.0092 0.0479 2.54 0.0189 0.8046 65.16 0.0123 Subject E 0.0262 2.20 0.0119 0.5840 69.12 0.0084 Subject F 0.0299 3.59 0.0083 0.4340 69.48 0.0062

Concentrations of 6β-OHF and cortisol and, 6β-OHF/cortisol ratio in DBS and plasma for 6 healthy subjects (n = 10)

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60 DBS 6β-OHF/cortisol CYP3A CYP3A DBS CYP3A CYP3A

Correlation between 6β-OHF/cortisol ratio in plasma (x-axis) and 6β-OHF/cortisol ratio in DBS (y-axis) Fig. 3-5 0.000 0.005 0.010 0.015 0.020 0.025 0.000 0.005 0.010 0.015 (n = 10) y r = 1.5447x - 0.0012 = 0.9548 6β -O H F /c or ti sol r at io i n D B S

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CYP3A 6β-hydroxycortisol

(6β-OHF)/cortisol 6β-OHF cortisol

LC-MS/MS CYP3A CYP3A cortisol 6β- (CLm(6β)) 6β-OHF/cortisol CYP3A 6β-OHF/cortisol 6β-OHF/cortisol CYP3A

(CLDTX) Dried blood spots (DBS)

6β-OHF/cortisol CYP3A 1 6β-hydroxycortisol cortisol LC-MS/MS (6β-OHF-2_H 4, cortisol-13C4) LC-MS/MS 6β-OHF cortisol ( ) 38.51 pg/mL (6β-OHF) 38.10 pg/mL (cortisol) (R.E.) 5.4% ~ 3.4% (R.S.D) 2.1% ~ 3.8% ( )

Electrospray ionization negative ion mode (ESI ( ))

38.51 pg/mL (6β-OHF) 38.10 pg/mL (cortisol) R.E. 4.90% ~ 0.19% R.S.D. 3.92% 6β-OHF/cortisol 6β-OHF cortisol 2 6β-hydroxycortisol/cortisol CYP3A 3 CLm(6β)

3 6β-OHF/cortisol CLm(6β)

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62

61%, 51%, 39% CLm(6β) (57%, 46%, 41%) 6β-OHF/cortisol CLm(6β) r = 0.9054

2 1.2 ~ 2.5 1 1 1.9 1.4 6β-OHF/cortisol CLm(6β) (1.5 ~ 3.0 ) 22 6β-OHF/cortisol 0.0093 ± 0.0030 (Mean ± S.D.) 0.0059 ~ 0.0172 20 6β-OHF/cortisol CLDTX 6β-OHF/cortisol CLDTX 1 6β-OHF/cortisol CYP3A

3 Dried blood spots 6β-hydroxycortisol/cortisol CYP3A DBS 6β-OHF/cortisol CYP3A 1 LC-MS/MS DBS 6β-OHF cortisol DBS 4 0.0125% 50 µL 40 µL LC-MS/MS 0.0051 pg (6β-OHF), 2.02 pg (cortisol)

R.E. 5.68% (6β-OHF), 1.89% (cortisol)

R.S.D. 5.29% 2 DBS

6β-OHF cortisol 6β-OHF cortisol

65% (6β-OHF), 85% (cortisol) 6 ( 3 , 3 ) DBS 6β-OHF/cortisol DBS 6β-OHF/cortisol DBS DBS 6β-OHF/cortisol 6β-OHF/cortisol 0.9548 DBS

CYP3A CLm(6β)

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63 CYP3A

1 6β-OHF/cortisol

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1) Sensitive and simultaneous quantitation of 6β-hydroxycortisol and cortisol in human plasma by LC-MS/MS coupled with stable isotope dilution method.

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