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Target	mRNA sequence	Primer/probe	Sequence
CYP1A2	NM_000761.3	5'-Primer	5'-AAA TGC TGT GTC TTC GTA AAC CAG-3'
		3'-Primer	5'-CGC TCA GGC CGG AAC TC-3'
		Probe	5'-(FAM) cca gag ctg tgg gag gac ccc tc (Tamra)-3'
CYP2B6	NM_000767.4	5'-Primer	5'-GCC ACC CTA ACA CCC ATG AC-3'
		3'-Primer	5'-TGA GTA GGC CTC TTC TAT CCA TCT G-3'
		Probe	5'-(FAM) cac cag ggc ccc gcc ctc t (Tamra)-3'
CYP3A4	NM_017460.5	5'-Primer	5'-GTG TGT TTC CAA GAG AAG TTA CAA ATT T-3'
		3'-Primer	5'-CCA CTC GGT GCT TTT GTG TAT C-3'
		Probe	5'-(FAM) cga ggc gac ttt ctt tca tcc ttt tta cag a (Tamra)-3'
β-Actin	NM_001101	5'-Primer	5'-GAC TAC CTC ATG AAG ATC CTC ACC-3'
		3'-Primer	5'-TCT CCT TAA TGT CAC GCA CGA TT-3'
		Probe	5'-(FAM) CGG CTA CAG CTT CAC CAC CAC GGC (TAMRA)-3'

Target

mRNA sequence

Primer/probe

Sequence

CYP1A2

NM_000761.3

5'-Primer

5'-AAA TGC TGT GTC TTC GTA AAC CAG-3'

3'-Primer

5'-CGC TCA GGC CGG AAC TC-3'

Probe

5'-(FAM) cca gag ctg tgg gag gac ccc tc (Tamra)-3'

CYP2B6

NM_000767.4

5'-Primer

5'-GCC ACC CTA ACA CCC ATG AC-3'

3'-Primer

5'-TGA GTA GGC CTC TTC TAT CCA TCT G-3'

Probe

5'-(FAM) cac cag ggc ccc gcc ctc t (Tamra)-3'

CYP3A4

NM_017460.5

5'-Primer

5'-GTG TGT TTC CAA GAG AAG TTA CAA ATT T-3'

3'-Primer

5'-CCA CTC GGT GCT TTT GTG TAT C-3'

Probe

5'-(FAM) cga ggc gac ttt ctt tca tcc ttt tta cag a (Tamra)-3'

β-Actin

NM_001101

5'-Primer

5'-GAC TAC CTC ATG AAG ATC CTC ACC-3'

3'-Primer

5'-TCT CCT TAA TGT CAC GCA CGA TT-3'

Probe

5'-(FAM) CGG CTA CAG CTT CAC CAC CAC GGC (TAMRA)-3'

Target	mRNA sequence	Primer/probe	Sequence
CYP1A2	NM_000761.3	5'-Primer	5'-AAA TGC TGT GTC TTC GTA AAC CAG-3'
		3'-Primer	5'-CGC TCA GGC CGG AAC TC-3'
		Probe	5'-(FAM) cca gag ctg tgg gag gac ccc tc (Tamra)-3'
CYP2B6	NM_000767.4	5'-Primer	5'-GCC ACC CTA ACA CCC ATG AC-3'
		3'-Primer	5'-TGA GTA GGC CTC TTC TAT CCA TCT G-3'
		Probe	5'-(FAM) cac cag ggc ccc gcc ctc t (Tamra)-3'
CYP3A4	NM_017460.5	5'-Primer	5'-GTG TGT TTC CAA GAG AAG TTA CAA ATT T-3'
		3'-Primer	5'-CCA CTC GGT GCT TTT GTG TAT C-3'
		Probe	5'-(FAM) cga ggc gac ttt ctt tca tcc ttt tta cag a (Tamra)-3'
β-Actin	NM_001101	5'-Primer	5'-GAC TAC CTC ATG AAG ATC CTC ACC-3'
		3'-Primer	5'-TCT CCT TAA TGT CAC GCA CGA TT-3'
		Probe	5'-(FAM) CGG CTA CAG CTT CAC CAC CAC GGC (TAMRA)-3'

Target

mRNA sequence

Primer/probe

Sequence

CYP1A2

NM_000761.3

5'-Primer

5'-AAA TGC TGT GTC TTC GTA AAC CAG-3'

3'-Primer

5'-CGC TCA GGC CGG AAC TC-3'

Probe

5'-(FAM) cca gag ctg tgg gag gac ccc tc (Tamra)-3'

CYP2B6

NM_000767.4

5'-Primer

5'-GCC ACC CTA ACA CCC ATG AC-3'

3'-Primer

5'-TGA GTA GGC CTC TTC TAT CCA TCT G-3'

Probe

5'-(FAM) cac cag ggc ccc gcc ctc t (Tamra)-3'

CYP3A4

NM_017460.5

5'-Primer

5'-GTG TGT TTC CAA GAG AAG TTA CAA ATT T-3'

3'-Primer

5'-CCA CTC GGT GCT TTT GTG TAT C-3'

Probe

5'-(FAM) cga ggc gac ttt ctt tca tcc ttt tta cag a (Tamra)-3'

β-Actin

NM_001101

5'-Primer

5'-GAC TAC CTC ATG AAG ATC CTC ACC-3'

3'-Primer

5'-TCT CCT TAA TGT CAC GCA CGA TT-3'

Probe

5'-(FAM) CGG CTA CAG CTT CAC CAC CAC GGC (TAMRA)-3'

CYP450 enzyme	Substrate	Incubation condition
CYP1A2	Phenacetin	0.250	10	5.00, 10.0, 30.0, 50.0, 100, 200, 400
CYP2B6	Bupropion	0.100	15	5.00, 10.0, 30.0, 50.0, 100, 200, 500
CYP2C8	Amodiaquine	0.100	10	0.300, 0.600, 1.00, 2.00, 5.00, 10.0, 20.0
CYP2C9	Diclofenac	0.250	10	1.00, 2.00, 5.00, 10.0, 50.0, 100, 200
CYP2C19	S-Mephenytoin	1.00	60	2.00, 5.00, 10.0, 30.0, 60.0, 100, 200
CYP2D6	Dextromethorphan	0.100	20	0.100, 0.500, 1.00, 5.00, 10.0, 20.0, 30.0
CYP3A4/5	Midazolam	0.100	5	0.200, 0.500, 1.00, 3.00, 10.0, 20.0, 50.0
CYP3A4/5	Testosterone	0.100	15	5.00, 10.0, 20.0, 50.0, 100.0, 200, 300

CYP450 enzyme

Substrate

Incubation condition

Enzyme activity

Protein concentration/mg·mL^-1

Time/min

Substrate concentration/μmol·L^-1

CYP1A2

Phenacetin

0.250

5.00, 10.0, 30.0, 50.0, 100, 200, 400

CYP2B6

Bupropion

0.100

5.00, 10.0, 30.0, 50.0, 100, 200, 500

CYP2C8

Amodiaquine

0.100

0.300, 0.600, 1.00, 2.00, 5.00, 10.0, 20.0

CYP2C9

Diclofenac

0.250

1.00, 2.00, 5.00, 10.0, 50.0, 100, 200

CYP2C19

S-Mephenytoin

1.00

2.00, 5.00, 10.0, 30.0, 60.0, 100, 200

CYP2D6

Dextromethorphan

0.100

0.100, 0.500, 1.00, 5.00, 10.0, 20.0, 30.0

CYP3A4/5

Midazolam

0.100

0.200, 0.500, 1.00, 3.00, 10.0, 20.0, 50.0

CYP3A4/5

Testosterone

0.100

5.00, 10.0, 20.0, 50.0, 100.0, 200, 300

CYP450 enzyme	Substrate	Incubation condition
CYP1A2	Phenacetin	0.250	10	5.00, 10.0, 30.0, 50.0, 100, 200, 400
CYP2B6	Bupropion	0.100	15	5.00, 10.0, 30.0, 50.0, 100, 200, 500
CYP2C8	Amodiaquine	0.100	10	0.300, 0.600, 1.00, 2.00, 5.00, 10.0, 20.0
CYP2C9	Diclofenac	0.250	10	1.00, 2.00, 5.00, 10.0, 50.0, 100, 200
CYP2C19	S-Mephenytoin	1.00	60	2.00, 5.00, 10.0, 30.0, 60.0, 100, 200
CYP2D6	Dextromethorphan	0.100	20	0.100, 0.500, 1.00, 5.00, 10.0, 20.0, 30.0
CYP3A4/5	Midazolam	0.100	5	0.200, 0.500, 1.00, 3.00, 10.0, 20.0, 50.0
CYP3A4/5	Testosterone	0.100	15	5.00, 10.0, 20.0, 50.0, 100.0, 200, 300

CYP450 enzyme

Substrate

Incubation condition

Enzyme activity

Protein concentration/mg·mL^-1

Time/min

Substrate concentration/μmol·L^-1

CYP1A2

Phenacetin

0.250

5.00, 10.0, 30.0, 50.0, 100, 200, 400

CYP2B6

Bupropion

0.100

5.00, 10.0, 30.0, 50.0, 100, 200, 500

CYP2C8

Amodiaquine

0.100

0.300, 0.600, 1.00, 2.00, 5.00, 10.0, 20.0

CYP2C9

Diclofenac

0.250

1.00, 2.00, 5.00, 10.0, 50.0, 100, 200

CYP2C19

S-Mephenytoin

1.00

2.00, 5.00, 10.0, 30.0, 60.0, 100, 200

CYP2D6

Dextromethorphan

0.100

0.100, 0.500, 1.00, 5.00, 10.0, 20.0, 30.0

CYP3A4/5

Midazolam

0.100

0.200, 0.500, 1.00, 3.00, 10.0, 20.0, 50.0

CYP3A4/5

Testosterone

0.100

5.00, 10.0, 20.0, 50.0, 100.0, 200, 300

CYP450 enzyme	Substrate	Substrate concentration /μmol·L^-1	Positive control of inhibitor	Positive control concentration/μmol·L^-1
CYP1A2	Phenacetin	150	α-Naphthoflavone	0, 0.00100, 0.00500, 0.0100, 0.0300, 0.0600, 0.100, 1.00, 10.0
CYP2B6	Bupropion	100	Thiotepa	0, 0.100, 0.500, 1.00, 5.00, 10.0, 20.0, 50.0, 100
CYP2C8	Amodiaquine	2.00	Quercetin	0, 0.0500, 0.100, 0.500, 1.00, 2.00, 5.00, 10.0, 20.0
CYP2C9	Diclofenac	10.0	Sulfaphenazole	0, 0.0100, 0.0500, 0.100, 0.300, 0.600, 1.00, 3.00, 10.0
CYP2C19	S-Mephenytoin	35.0	Ticlopidine	0, 0.00500, 0.0500, 0.500, 2.00, 5.00, 10.0, 20.0, 50.0
CYP2D6	Dextromethorphan	10.0	Quinidine	0, 0.100, 0.200, 0.500, 1.00, 2.00, 5.00, 10.0, 20.0
CYP3A4/5	Midazolam	2.00	Ketoconazole	0, 0.00100, 0.0100, 0.0500, 0.100, 0.500, 1.00, 5.00, 10.0
CYP3A4/5	Testosterone	50.0	Ketoconazole	0, 0.000100, 0.000500, 0.00100, 0.00500, 0.0100, 0.100, 1.00, 5.00

CYP450 enzyme

Substrate

Substrate concentration /μmol·L^-1

Positive control of inhibitor

Positive control concentration/μmol·L^-1

CYP1A2

Phenacetin

150

α-Naphthoflavone

0, 0.00100, 0.00500, 0.0100, 0.0300, 0.0600, 0.100, 1.00, 10.0

CYP2B6

Bupropion

100

Thiotepa

0, 0.100, 0.500, 1.00, 5.00, 10.0, 20.0, 50.0, 100

CYP2C8

Amodiaquine

2.00

Quercetin

0, 0.0500, 0.100, 0.500, 1.00, 2.00, 5.00, 10.0, 20.0

CYP2C9

Diclofenac

10.0

Sulfaphenazole

0, 0.0100, 0.0500, 0.100, 0.300, 0.600, 1.00, 3.00, 10.0

CYP2C19

S-Mephenytoin

35.0

Ticlopidine

0, 0.00500, 0.0500, 0.500, 2.00, 5.00, 10.0, 20.0, 50.0

CYP2D6

Dextromethorphan

10.0

Quinidine

0, 0.100, 0.200, 0.500, 1.00, 2.00, 5.00, 10.0, 20.0

CYP3A4/5

Midazolam

2.00

Ketoconazole

0, 0.00100, 0.0100, 0.0500, 0.100, 0.500, 1.00, 5.00, 10.0

CYP3A4/5

Testosterone

50.0

Ketoconazole

0, 0.000100, 0.000500, 0.00100, 0.00500, 0.0100, 0.100, 1.00, 5.00

CYP450 enzyme	Substrate	Substrate concentration /μmol·L^-1	Positive control of inhibitor	Positive control concentration/μmol·L^-1
CYP1A2	Phenacetin	150	α-Naphthoflavone	0, 0.00100, 0.00500, 0.0100, 0.0300, 0.0600, 0.100, 1.00, 10.0
CYP2B6	Bupropion	100	Thiotepa	0, 0.100, 0.500, 1.00, 5.00, 10.0, 20.0, 50.0, 100
CYP2C8	Amodiaquine	2.00	Quercetin	0, 0.0500, 0.100, 0.500, 1.00, 2.00, 5.00, 10.0, 20.0
CYP2C9	Diclofenac	10.0	Sulfaphenazole	0, 0.0100, 0.0500, 0.100, 0.300, 0.600, 1.00, 3.00, 10.0
CYP2C19	S-Mephenytoin	35.0	Ticlopidine	0, 0.00500, 0.0500, 0.500, 2.00, 5.00, 10.0, 20.0, 50.0
CYP2D6	Dextromethorphan	10.0	Quinidine	0, 0.100, 0.200, 0.500, 1.00, 2.00, 5.00, 10.0, 20.0
CYP3A4/5	Midazolam	2.00	Ketoconazole	0, 0.00100, 0.0100, 0.0500, 0.100, 0.500, 1.00, 5.00, 10.0
CYP3A4/5	Testosterone	50.0	Ketoconazole	0, 0.000100, 0.000500, 0.00100, 0.00500, 0.0100, 0.100, 1.00, 5.00

CYP450 enzyme

Substrate

Substrate concentration /μmol·L^-1

Positive control of inhibitor

Positive control concentration/μmol·L^-1

CYP1A2

Phenacetin

150

α-Naphthoflavone

0, 0.00100, 0.00500, 0.0100, 0.0300, 0.0600, 0.100, 1.00, 10.0

CYP2B6

Bupropion

100

Thiotepa

0, 0.100, 0.500, 1.00, 5.00, 10.0, 20.0, 50.0, 100

CYP2C8

Amodiaquine

2.00

Quercetin

0, 0.0500, 0.100, 0.500, 1.00, 2.00, 5.00, 10.0, 20.0

CYP2C9

Diclofenac

10.0

Sulfaphenazole

0, 0.0100, 0.0500, 0.100, 0.300, 0.600, 1.00, 3.00, 10.0

CYP2C19

S-Mephenytoin

35.0

Ticlopidine

0, 0.00500, 0.0500, 0.500, 2.00, 5.00, 10.0, 20.0, 50.0

CYP2D6

Dextromethorphan

10.0

Quinidine

0, 0.100, 0.200, 0.500, 1.00, 2.00, 5.00, 10.0, 20.0

CYP3A4/5

Midazolam

2.00

Ketoconazole

0, 0.00100, 0.0100, 0.0500, 0.100, 0.500, 1.00, 5.00, 10.0

CYP3A4/5

Testosterone

50.0

Ketoconazole

0, 0.000100, 0.000500, 0.00100, 0.00500, 0.0100, 0.100, 1.00, 5.00

Study	CYP450 enzyme	Analyte	MRM transition	DP/V	EP/V	CE/V	CXP/V
Inhibition	CYP1A2	Acetaminophen	152.2 → 110.1	100	10	21	12
	CYP2B6	Hydroxybupropion	256.1 → 238.1	65	7	20	18
	CYP2C8	N-Desethyl amodiaquine	328.2 → 283.2	120	10	26	22
	CYP2C9	4'-Hydroxydiclofenac	312.1 → 266.1	100	10	17.5	20
	CYP2C19	4'-Hydroxymephenytoin	235.2 → 150.1	120	10	25	20
	CYP2D6	Dextrorphan	258.3 → 157.1	130	10	47	13
	CYP3A4/5	1'-Hydroxymidazolam	305.3 → 269.3	120	12	18	18
	CYP3A4/5	6β-Hydroxytestosterone	342.1 → 324.1	120	10	28.5	22
	IS	Ornidazole	220.1 → 128.0	120	10	20	13
Induction	CYP1A2	Acetaminophen	152.2 → 110.1	120	10	21	12
	CYP2B6	Hydroxybupropion	256.1 → 238.1	120	10	20	18
	CYP3A4/5	6β-Hydroxytestosterone	305.1 → 324.1	120	10	18	18
	IS	Ornidazole	220.1 → 128.0	120	10	20	13

Study

CYP450 enzyme

Analyte

MRM transition

DP/V

EP/V

CE/V

CXP/V

Inhibition

CYP1A2

Acetaminophen

152.2 → 110.1

100

CYP2B6

Hydroxybupropion

256.1 → 238.1

CYP2C8

N-Desethyl amodiaquine

328.2 → 283.2

120

CYP2C9

4'-Hydroxydiclofenac

312.1 → 266.1

100

17.5

CYP2C19

4'-Hydroxymephenytoin

235.2 → 150.1

120

CYP2D6

Dextrorphan

258.3 → 157.1

130

CYP3A4/5

1'-Hydroxymidazolam

305.3 → 269.3

120

CYP3A4/5

6β-Hydroxytestosterone

342.1 → 324.1

120

28.5

Ornidazole

220.1 → 128.0

120

Induction

CYP1A2

Acetaminophen

152.2 → 110.1

120

CYP2B6

Hydroxybupropion

256.1 → 238.1

120

CYP3A4/5

6β-Hydroxytestosterone

305.1 → 324.1

120

Ornidazole

220.1 → 128.0

120

Study	CYP450 enzyme	Analyte	MRM transition	DP/V	EP/V	CE/V	CXP/V
Inhibition	CYP1A2	Acetaminophen	152.2 → 110.1	100	10	21	12
	CYP2B6	Hydroxybupropion	256.1 → 238.1	65	7	20	18
	CYP2C8	N-Desethyl amodiaquine	328.2 → 283.2	120	10	26	22
	CYP2C9	4'-Hydroxydiclofenac	312.1 → 266.1	100	10	17.5	20
	CYP2C19	4'-Hydroxymephenytoin	235.2 → 150.1	120	10	25	20
	CYP2D6	Dextrorphan	258.3 → 157.1	130	10	47	13
	CYP3A4/5	1'-Hydroxymidazolam	305.3 → 269.3	120	12	18	18
	CYP3A4/5	6β-Hydroxytestosterone	342.1 → 324.1	120	10	28.5	22
	IS	Ornidazole	220.1 → 128.0	120	10	20	13
Induction	CYP1A2	Acetaminophen	152.2 → 110.1	120	10	21	12
	CYP2B6	Hydroxybupropion	256.1 → 238.1	120	10	20	18
	CYP3A4/5	6β-Hydroxytestosterone	305.1 → 324.1	120	10	18	18
	IS	Ornidazole	220.1 → 128.0	120	10	20	13

Study

CYP450 enzyme

Analyte

MRM transition

DP/V

EP/V

CE/V

CXP/V

Inhibition

CYP1A2

Acetaminophen

152.2 → 110.1

100

CYP2B6

Hydroxybupropion

256.1 → 238.1

CYP2C8

N-Desethyl amodiaquine

328.2 → 283.2

120

CYP2C9

4'-Hydroxydiclofenac

312.1 → 266.1

100

17.5

CYP2C19

4'-Hydroxymephenytoin

235.2 → 150.1

120

CYP2D6

Dextrorphan

258.3 → 157.1

130

CYP3A4/5

1'-Hydroxymidazolam

305.3 → 269.3

120

CYP3A4/5

6β-Hydroxytestosterone

342.1 → 324.1

120

28.5

Ornidazole

220.1 → 128.0

120

Induction

CYP1A2

Acetaminophen

152.2 → 110.1

120

CYP2B6

Hydroxybupropion

256.1 → 238.1

120

CYP3A4/5

6β-Hydroxytestosterone

305.1 → 324.1

120

Ornidazole

220.1 → 128.0

120

Study	Analyte	SC range/nmol·L^-1	QC level/nmol·L^-1	Typical standard curve
Inhibition	Acetaminophen	25.0-2 500	60.0, 300, 2 000	ƒ = 4.65e^-5 × C -1.18e^-4, r = 0.999 0
	Hydroxybupropion	10.0-1 000	24.0, 120, 800	ƒ = 1.75e^-4 × C -2.22e^-4, r = 0.999 7
	N-Desethyl amodiaquine	5.00-500	12.0, 60.0, 400	ƒ = 2.90e^-4 × C -9.77e^-5, r = 0.999 9
	4'-Hydroxydiclofenac	250-25 000	600, 3 000, 20 000	ƒ = 5.04e^-5 × C + 2.16e^-4, r = 0.999 9
	4'-Hydroxymephenytoin	100-10 000	240, 1 200, 8 000	ƒ = 2.16e^-5 × C -1.75e^-5, r = 0.999 7
	Dextrorphan	5.00-500	12.0, 60.0, 400	ƒ = 1.34e^-4 × C -9.90e^-5, r = 0.999 9
	1'-Hydroxymidazolam	5.00-500	12.0, 60.0, 400	ƒ = 4.19e^-4× C -1.99e^-4, r = 0.999 3
	6β-Hydroxytestosterone	100-10 000	240, 1 200, 8 000	ƒ = 2.72e^-5 × C -5.05e^-5, r = 0.999 8
Induction	Acetaminophen	100-40 000	250, 3 000, 32 000	ƒ = 6.13e^-2 × C + -5.41e^-4, r = 0.999 2
	Hydroxybupropion	100-40 000	250, 3 000, 32 000	ƒ = 2.41e^-1 × C + 1.25 e^-3, r = 0.997 6
	6β-Hydroxytestosterone	100-40 000	250, 3 000, 32 000	ƒ = 3.61e^-2 × C -3.67e^-4, r = 0.999 0

Study

Analyte

SC range/nmol·L^-1

QC level/nmol·L^-1

Typical standard curve

Inhibition

Acetaminophen

25.0-2 500

60.0, 300, 2 000

ƒ = 4.65e^-5 × C -1.18e^-4, r = 0.999 0

Hydroxybupropion

10.0-1 000

24.0, 120, 800

ƒ = 1.75e^-4 × C -2.22e^-4, r = 0.999 7

N-Desethyl amodiaquine

5.00-500

12.0, 60.0, 400

ƒ = 2.90e^-4 × C -9.77e^-5, r = 0.999 9

4'-Hydroxydiclofenac

250-25 000

600, 3 000, 20 000

ƒ = 5.04e^-5 × C + 2.16e^-4, r = 0.999 9

4'-Hydroxymephenytoin

100-10 000

240, 1 200, 8 000

ƒ = 2.16e^-5 × C -1.75e^-5, r = 0.999 7

Dextrorphan

5.00-500

12.0, 60.0, 400

ƒ = 1.34e^-4 × C -9.90e^-5, r = 0.999 9

1'-Hydroxymidazolam

5.00-500

12.0, 60.0, 400

ƒ = 4.19e^-4× C -1.99e^-4, r = 0.999 3

6β-Hydroxytestosterone

100-10 000

240, 1 200, 8 000

ƒ = 2.72e^-5 × C -5.05e^-5, r = 0.999 8

Induction

Acetaminophen

100-40 000

250, 3 000, 32 000

ƒ = 6.13e^-2 × C + -5.41e^-4, r = 0.999 2

Hydroxybupropion

100-40 000

250, 3 000, 32 000

ƒ = 2.41e^-1 × C + 1.25 e^-3, r = 0.997 6

6β-Hydroxytestosterone

100-40 000

250, 3 000, 32 000

ƒ = 3.61e^-2 × C -3.67e^-4, r = 0.999 0

Study	Analyte	SC range/nmol·L^-1	QC level/nmol·L^-1	Typical standard curve
Inhibition	Acetaminophen	25.0-2 500	60.0, 300, 2 000	ƒ = 4.65e^-5 × C -1.18e^-4, r = 0.999 0
	Hydroxybupropion	10.0-1 000	24.0, 120, 800	ƒ = 1.75e^-4 × C -2.22e^-4, r = 0.999 7
	N-Desethyl amodiaquine	5.00-500	12.0, 60.0, 400	ƒ = 2.90e^-4 × C -9.77e^-5, r = 0.999 9
	4'-Hydroxydiclofenac	250-25 000	600, 3 000, 20 000	ƒ = 5.04e^-5 × C + 2.16e^-4, r = 0.999 9
	4'-Hydroxymephenytoin	100-10 000	240, 1 200, 8 000	ƒ = 2.16e^-5 × C -1.75e^-5, r = 0.999 7
	Dextrorphan	5.00-500	12.0, 60.0, 400	ƒ = 1.34e^-4 × C -9.90e^-5, r = 0.999 9
	1'-Hydroxymidazolam	5.00-500	12.0, 60.0, 400	ƒ = 4.19e^-4× C -1.99e^-4, r = 0.999 3
	6β-Hydroxytestosterone	100-10 000	240, 1 200, 8 000	ƒ = 2.72e^-5 × C -5.05e^-5, r = 0.999 8
Induction	Acetaminophen	100-40 000	250, 3 000, 32 000	ƒ = 6.13e^-2 × C + -5.41e^-4, r = 0.999 2
	Hydroxybupropion	100-40 000	250, 3 000, 32 000	ƒ = 2.41e^-1 × C + 1.25 e^-3, r = 0.997 6
	6β-Hydroxytestosterone	100-40 000	250, 3 000, 32 000	ƒ = 3.61e^-2 × C -3.67e^-4, r = 0.999 0

Study

Analyte

SC range/nmol·L^-1

QC level/nmol·L^-1

Typical standard curve

Inhibition

Acetaminophen

25.0-2 500

60.0, 300, 2 000

ƒ = 4.65e^-5 × C -1.18e^-4, r = 0.999 0

Hydroxybupropion

10.0-1 000

24.0, 120, 800

ƒ = 1.75e^-4 × C -2.22e^-4, r = 0.999 7

N-Desethyl amodiaquine

5.00-500

12.0, 60.0, 400

ƒ = 2.90e^-4 × C -9.77e^-5, r = 0.999 9

4'-Hydroxydiclofenac

250-25 000

600, 3 000, 20 000

ƒ = 5.04e^-5 × C + 2.16e^-4, r = 0.999 9

4'-Hydroxymephenytoin

100-10 000

240, 1 200, 8 000

ƒ = 2.16e^-5 × C -1.75e^-5, r = 0.999 7

Dextrorphan

5.00-500

12.0, 60.0, 400

ƒ = 1.34e^-4 × C -9.90e^-5, r = 0.999 9

1'-Hydroxymidazolam

5.00-500

12.0, 60.0, 400

ƒ = 4.19e^-4× C -1.99e^-4, r = 0.999 3

6β-Hydroxytestosterone

100-10 000

240, 1 200, 8 000

ƒ = 2.72e^-5 × C -5.05e^-5, r = 0.999 8

Induction

Acetaminophen

100-40 000

250, 3 000, 32 000

ƒ = 6.13e^-2 × C + -5.41e^-4, r = 0.999 2

Hydroxybupropion

100-40 000

250, 3 000, 32 000

ƒ = 2.41e^-1 × C + 1.25 e^-3, r = 0.997 6

6β-Hydroxytestosterone

100-40 000

250, 3 000, 32 000

ƒ = 3.61e^-2 × C -3.67e^-4, r = 0.999 0

CYP450 enzyme	Substrate	Positive control	K_m/μmol·L^-1	IC₅₀/μmol·L^-1
CYP1A2	Phenacetin	α-Naphthoflavone	114 ± 33	1.7-152	0.022 0 ± 0.012 3
CYP2B6	Bupropion	Thiotepa	158 ± 108	67-168	0.444 ± 0.068
CYP2C8	Amodiaquine	Quercetin	2.61 ± 0.06	2.4	1.55 ± 0.69
CYP2C9	Diclofenac	Sulfaphenazole	9.16 ± 1.17	3.4-52	0.966 ± 0.348
CYP2C19	S-Mephenytoin	Ticlopidine	16.6 ± 6.6	13-35	0.223 ± 0.106
CYP2D6	Dextromethorphan	Quinidine	20.2 ± 12.8	0.44-8.5	0.685 ± 0.266
CYP3A4/5	Midazolam	Ketoconazole	1.60 ± 0.34	1.0-14	0.773 ± 0.178
CYP3A4/5	Testosterone	Ketoconazole	78.1 ± 4.7	52-94	0.968 ± 0.144

CYP450 enzyme

Substrate

Positive control

K_m/μmol·L^-1

IC₅₀/μmol·L^-1

Result

FDA^{[8, 9]}

CYP1A2

Phenacetin

α-Naphthoflavone

114 ± 33

1.7-152

0.022 0 ± 0.012 3

CYP2B6

Bupropion

Thiotepa

158 ± 108

67-168

0.444 ± 0.068

CYP2C8

Amodiaquine

Quercetin

2.61 ± 0.06

2.4

1.55 ± 0.69

CYP2C9

Diclofenac

Sulfaphenazole

9.16 ± 1.17

3.4-52

0.966 ± 0.348

CYP2C19

S-Mephenytoin

Ticlopidine

16.6 ± 6.6

13-35

0.223 ± 0.106

CYP2D6

Dextromethorphan

Quinidine

20.2 ± 12.8

0.44-8.5

0.685 ± 0.266

CYP3A4/5

Midazolam

Ketoconazole

1.60 ± 0.34

1.0-14

0.773 ± 0.178

CYP3A4/5

Testosterone

Ketoconazole

78.1 ± 4.7

52-94

0.968 ± 0.144

CYP450 enzyme	Substrate	Positive control	K_m/μmol·L^-1	IC₅₀/μmol·L^-1
CYP1A2	Phenacetin	α-Naphthoflavone	114 ± 33	1.7-152	0.022 0 ± 0.012 3
CYP2B6	Bupropion	Thiotepa	158 ± 108	67-168	0.444 ± 0.068
CYP2C8	Amodiaquine	Quercetin	2.61 ± 0.06	2.4	1.55 ± 0.69
CYP2C9	Diclofenac	Sulfaphenazole	9.16 ± 1.17	3.4-52	0.966 ± 0.348
CYP2C19	S-Mephenytoin	Ticlopidine	16.6 ± 6.6	13-35	0.223 ± 0.106
CYP2D6	Dextromethorphan	Quinidine	20.2 ± 12.8	0.44-8.5	0.685 ± 0.266
CYP3A4/5	Midazolam	Ketoconazole	1.60 ± 0.34	1.0-14	0.773 ± 0.178
CYP3A4/5	Testosterone	Ketoconazole	78.1 ± 4.7	52-94	0.968 ± 0.144

CYP450 enzyme

Substrate

Positive control

K_m/μmol·L^-1

IC₅₀/μmol·L^-1

Result

FDA^{[8, 9]}

CYP1A2

Phenacetin

α-Naphthoflavone

114 ± 33

1.7-152

0.022 0 ± 0.012 3

CYP2B6

Bupropion

Thiotepa

158 ± 108

67-168

0.444 ± 0.068

CYP2C8

Amodiaquine

Quercetin

2.61 ± 0.06

2.4

1.55 ± 0.69

CYP2C9

Diclofenac

Sulfaphenazole

9.16 ± 1.17

3.4-52

0.966 ± 0.348

CYP2C19

S-Mephenytoin

Ticlopidine

16.6 ± 6.6

13-35

0.223 ± 0.106

CYP2D6

Dextromethorphan

Quinidine

20.2 ± 12.8

0.44-8.5

0.685 ± 0.266

CYP3A4/5

Midazolam

Ketoconazole

1.60 ± 0.34

1.0-14

0.773 ± 0.178

CYP3A4/5

Testosterone

Ketoconazole

78.1 ± 4.7

52-94

0.968 ± 0.144

药物对人肝CYP450酶诱导和抑制作用体外评价体系的建立与验证

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孙鲁宁 ¹^,^† , 吴春勇 ²^,³^,^† , 赵舜波 ⁴ , 刘瑞娟 ³ , 王雷 ³ , 丁黎 ²^,³^,⁴^,^* , 王永庆 ¹^,^*

药学学报 | 研究论文 2017,52(12): 1924-1932

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药学学报 | 研究论文 2017, 52(12): 1924-1932

药物对人肝CYP450酶诱导和抑制作用体外评价体系的建立与验证

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孙鲁宁¹^,^†, 吴春勇²^,³^,^†, 赵舜波⁴, 刘瑞娟³, 王雷³, 丁黎²^,³^,⁴^,^*, 王永庆¹^,^*

作者信息

1.南京医科大学第一附属医院, 江苏南京 210029

2.中国药科大学药物质量与安全预警教育部重点实验室, 江苏南京 210009

3.中国药科大学药物分析系, 江苏南京 210009

4.南京科利泰医药科技有限公司, 江苏南京 211100

通讯作者:

* 丁黎, Tel:86-25-83271289, E-mail:dinglihg@sina.com;

王永庆, Tel:86-25-68136984, E-mail:wyqjsph@163.com

Establishment of in vitro methods for evaluation of induction and inhibition of human CYP450 enzymes by drugs

Lu-ning SUN¹, Chun-yong WU²^,³, Shun-bo ZHAO⁴, Rui-juan LIU³, Lei WANG³, Li DING²^,³^,⁴^,^*, Yong-qing WANG¹^,^*

Affiliations

1. First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China

2. Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, Nanjing 210009, China

3. Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China

4. Nanjing Clinical Tech. Laboratories Inc., Nanjing 211100, China

出版时间: 2017-12-12 doi: 10.16438/j.0513-4870.2017-0700

文章导航

摘要

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建立药物对CYP450酶诱导与抑制作用的体外评价方法并进行验证。LC-MS/MS法测定人肝微粒体CYP450酶7种主要亚型的8个底物代谢产物；在优化的温孵条件下，底物法测定人肝微粒体CYP450酶的活性；应用已知抑制剂对所建立的温孵体系进行验证。LC-MS/MS法测定人肝细胞CYP450酶3种主要亚型的底物代谢产物；底物法测定人原代肝细胞CYP450酶的活性，同时收集温孵后的人肝细胞，应用Taqman荧光探针法测定主要亚型mRNA的表达量；应用已知诱导剂对所建立的温孵体系进行验证。建立的LC-MS/MS分析方法符合生物分析要求；已知抑制剂和诱导剂在本实验温孵体系下均表现出明显的抑制作用或诱导作用。建立的实验方法准确、可靠，可用于体外评价药物对CYP450酶的诱导或抑制作用。

关键词

人肝微粒体 / 人原代肝细胞 / CYP450酶 / 诱导作用 / 抑制作用 / 探针底物法 / LC-MS/MS / 药物相互作用

Abstract

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The aim of this study is to establish the in vitro methods for the study of induction and inhibition on CYP450 by drugs, and to validate the analytical method and incubation system. A method for the simultaneous determination of eight metabolites of seven subtypes of CYP450 enzymes probe substrates in human liver microsomes (HLM) was established and validated. The incubation system was optimized to confirm the incubation time and protein concentration of HLM, the enzyme activity of seven subtypes of CYP450 enzymes in HLM was determined, and the inhibition effects on each CYP450s were checked by positive controls. The method for the simultaneous determination of three metabolites of subtypes of CYP450 enzymes was established and validated in human primary cultured hepatocytes (HPCH) using the incubation medium. The enzyme activity of three subtypes of CYP450 enzymes in HPCH was determined, and the total RNA was extracted from HPCH after incubation. The expression of CYP450 enzymes were measured by Taqman fluorescence probe method. The induction effects on each CYP450s were examined using the positive controls. The established methods for the determination of metabolites of probe substrates were fully validated, and the results were conformed to the requirements of bioanalytical method validation. The induction and inhibition effects on each CYP450s were checked by positive controls. The established in vitro methods for the study of drug induction and inhibition on CYP450 were simple and reliable, which could be used in the investigation of enzyme induction or inhibition properties of new drug candidates and to evaluation the metabolic interactions of concomitant medication in clinical.

Key words

human liver microsome / human primary cultured hepatocytes / CYP450 / induction effect / inhibition effect / probe substrates method / LC-MS/MS / drug-drug interaction

引用本文

孙鲁宁, 吴春勇, 赵舜波, 刘瑞娟, 王雷, 丁黎, 王永庆. 药物对人肝CYP450酶诱导和抑制作用体外评价体系的建立与验证. 药学学报, 2017 , 52 (12) : 1924 -1932 . DOI: 10.16438/j.0513-4870.2017-0700

Lu-ning SUN, Chun-yong WU, Shun-bo ZHAO, Rui-juan LIU, Lei WANG, Li DING, Yong-qing WANG. Establishment of in vitro methods for evaluation of induction and inhibition of human CYP450 enzymes by drugs[J]. Acta Pharmaceutica Sinica, 2017 , 52 (12) : 1924 -1932 . DOI: 10.16438/j.0513-4870.2017-0700

正文

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细胞色素P450酶(CYP450)是微粒体混合功能氧化酶系中最重要的氧化酶, 临床上超过90%的药物由CYP450代谢^[1]。CYP450酶诱导和抑制所致的代谢性药物相互作用有可能显著改变合用药物的药动学、药效及毒性, 因此阐明药物对CYP450酶的诱导和抑制作用是评价药物安全性和有效性的重要组成部分^{[2, 3]}。不同实验室研究药物对CYP450酶诱导和抑制作用的实验结果相差较大, 需要建立可靠的方法以支持实验结果^{[4, 5]}。不仅需要对分析方法进行系统的方法学验证, 也需对实验体系(包括温孵条件、酶的活性和阳性对照的诱导和抑制能力)进行充分的考察^[6]。根据国家食品药品监督管理总局(CFDA)^[7]和美国食品药品监督管理局(FDA)颁布的药物相互作用研究相关指导原则^{[8, 9]}, 本研究建立药物对CYP450酶诱导和抑制作用体外评价方法并进行了验证。

本研究包括两方面内容: ①为研究药物对人肝微粒体CYP450酶主要亚型的抑制作用, 建立并验证测定人肝微粒体CYP450酶7种主要亚型的8个特异性底物代谢产物的LC-MS/MS法, 进而对温孵体系进行优化, 并采用已知抑制剂对体外系统进行验证; ②为研究药物对人肝细胞CYP450酶的诱导作用, 建立并验证测定人原代肝细胞CYP450酶3种主要亚型CYP1A2、CYP2B6和CYP3A4特异性底物代谢产物的LC-MS/MS法, 通过测定温孵液中3个特异性底物代谢产物的生成速率计算CYP450酶活性, 同时收集温孵后的人肝细胞提取总RNA, 采用实时荧光定量PCR法测定CYP1A2、CYP2B6和CYP3A4 mRNA表达量, 分别以酶活性及mRNA转录水平为终点, 采用已知诱导剂对体外系统进行验证。

材料与方法

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试剂与仪器 非那西丁、α-萘黄酮、奎尼丁、酮康唑和利福平购自TCI公司; 盐酸安非他酮、氢溴酸右美沙芬、马来酸咪达唑仑、对乙酰氨基酚、苯巴比妥和奥硝唑(内标)购自中国食品药品检定研究院; 盐酸阿莫地喹为USP对照品; 双氯芬酸钠、羟化安非他酮、4'-羟基双氯芬酸、右啡烷、6β-羟基睾酮、塞替派、槲皮素、盐酸噻氯匹定和Trizma碱购自Sigma公司; S-美芬妥英和奥美拉唑购自上海百灵威化学有限公司; 睾酮和磺胺苯吡唑购自Dr. Ehrenstorfer GmbH公司; N-去乙基阿莫地喹、4'-羟基美芬妥英和1-羟基咪达唑仑购自TRC公司; 还原型辅酶(NADPH)购自Roche Diagnostic GmbH公司; DEPC水购自碧云天生物技术研究所; 台盼蓝溶液购自南京凯基生物科技公司; RNAiso Plus、PrimeScript^TM RT reagent kit with gDNA Eraser和Premix EX Taq (Probe qPCR)购自大连Takara公司; 抗生素溶液、细胞培养液和细胞贴壁液购自瑞德肝脏疾病研究(上海)有限公司; 鼠尾胶原溶液购自康宁公司; 目的基因CYP1A2、CYP2B6、CYP3A4和内参β-actin的引物及Taqman探针(表 1)由大连Takara公司合成; 色谱纯甲醇和乙腈购自Merck公司, 其他溶剂和试剂均为分析纯或以上。

BP-211D型电子天平(德国Sartorius公司); 液质联用仪(液相色谱包括LC-20AD泵、SIL-20AC自动进样器和CTO-20AC柱温箱, 岛津(中国)有限公司; 质谱为QTRAP 5500, 美国应用生物公司; Analyst^TM色谱工作站, 1.6.1版本, 美国应用生物公司); Milli-Q Grandient纯化水仪(Millipore中国有限公司); HFsafe-900LC超净工作台(上海力申科学仪器有限公司); IX73-F22FL/PH倒置显微镜(奥林巴斯中国有限公司); CO₂/O₂培养箱(HF100, 上海力申科学仪器有限公司); StepOnePlus^TM Real-Time荧光定量PCR仪和Veriti PCR仪(美国应用生物公司); NANO DROP 2000微量紫外分光光度计(Thermo Scientific公司); Biotek-Eon酶标仪(美国伯腾仪器有限公司)。

人肝微粒体和人原代肝细胞 25例供体混合人肝微粒体(批号ZOF)和3个不同来源的人肝细胞(批号LTG、ONO和YOW)购自瑞德肝脏疾病研究(上海)有限公司, 人源材料经美国Bioreclamation IVT伦理委员会批准。

人肝微粒体的体外抑制研究

人肝微粒体温孵体系的确定分别将不同蛋白浓度的人肝微粒体与底物温孵, 加入NADPH启动反应后, 分别于温孵过程的不同时间点(CYP3A4/5: 0、3、5、10、15和20 min, 其余CYP450s: 0、5、10、15、20和30 min), 取出温孵溶液, 加入含内标的甲醇终止反应, 采用HPLC-MS/MS法测定底物代谢产物的生成量。在不同蛋白浓度下, 分别作温孵时间与底物代谢物生成量的线性回归方程, 选择温孵时间与底物代谢物生产量在线性范围内的微粒体蛋白浓度和温孵时间作为温孵条件, CYP450s各亚型的温孵条件包括微粒体蛋白浓度和温孵时间见表 2。

人肝微粒体CYP450s酶活性的评价于2.0 mL离心管中依次加入浓度为0.1 mol·L^-1 Tris-HCl缓冲液(pH 7.4) 100 μL、探针底物溶液(底物及其浓度范围见表 2) 2.00 μL和人肝微粒体(其中含MgCl₂的浓度为10.0 mmol·L^-1, 微粒体蛋白浓度见表 2) 80.0 μL后, 37 ℃预孵10 min, 再加入NADPH (10.0 mmol·L^-1, 温孵体系终浓度为1.0 mmol·L^-1) 20.0 μL启动反应, 一定时间(温孵时间见表 2)后, 加入含内标奥硝唑(2.00 μmol·L^-1)的甲醇溶液400 μL终止反应, 涡旋3 min, 16 000 r·min^-1离心10 min (4 ℃)。取CYP1A2、CYP2B6、CYP2C8、CYP2D6和CYP3A4/5微粒体处理后上清液各100 μL, 于同一1.5 mL离心管涡旋混匀, 取上清液100 μL, 再分别加入CYP2C9微粒体处理后上清液和CYP2C19微粒体处理后上清液各100 μL, 混匀, 进行LC-MS/MS分析, 测定温孵体系中生成的各底物相应的代谢产物浓度, 按下式计算各代谢产物的生成速率:代谢产物的生成速率=代谢产物的浓度/ (微粒体蛋白浓度×温孵反应时间)。使用GraphPad Prism 5.01软件按照非线性回归计算各底物的米氏常数K_m。

已知抑制剂半数抑制浓度的测定将不同浓度的已知抑制剂(2.00 μL抑制剂溶液阳性对照)分别与底物探针共同温孵, 底物和阳性对照抑制剂的浓度见表 3, 对照组(不含抑制剂)加入等体积的缓冲液, 余下按“人肝微粒体CYP450s酶活性的评价”项下操作。按下式计算剩余率:剩余率=生成速率(含抑制剂) /生成速率(不含抑制剂) × 100%。以剩余率为纵坐标, 抑制剂浓度的对数值为横坐标, 绘制抑制曲线, 使用GraphPad Prism 5.01软件按照非线性回归, 计算各抑制剂的半数抑制浓度(50% inhibition concentration, IC₅₀)。

人原代肝细胞的体外诱导研究

人原代肝细胞的培养细胞于37 ℃水浴解冻并转移至有细胞贴壁液的离心管中混悬, 用台盼蓝排斥实验检测活细胞数, 活细胞数 > 80%为合格。调整活细胞的密度至每毫升7.0×10⁵个活细胞, 按活细胞密度为1.4×10⁵个/孔接种到经胶原包被处理的48孔细胞培养板中, 在37 ℃和5% CO₂的条件下培养。第2天, 更换细胞贴壁液并洗去没有贴壁的细胞。第3天, 吸出每孔中的细胞贴壁液, 分别加入溶剂对照和诱导剂工作液200 μL, 每个条件3个复孔。第4天, 重复第3天的操作。第5天, 每孔测定CYP450s酶活性后, 剩余的细胞立即加入细胞培养液200 μL, 进行下一步的RNA提取。

人原代肝细胞CYP450s酶活性的测定于实验第5天, 移除每孔中的培养液, 分别加入特异性底物工作液200 μL, 37 ℃温孵1 h后, 从相应的孔中转移温孵液200 μL至1.5 mL离心管中, 涡旋混匀, 储存在-40 ℃冰箱中, 于200 μL温孵液样本中加入含内标奥硝唑(2.00 μmol·L^-1)的甲醇溶液600 μL, 涡旋3 min, 16 000 r·min^-1离心10 min (4 ℃)。分别取3种CYP450s亚型的反应温孵液处理后上清液各100 μL, 置于同一离心管中, 涡旋混匀, 进行LC-MS/MS分析, 通过测定特异性底物的代谢产物的生成速率(代谢物的生成速率=代谢物的生成浓度×培养液体积/温孵时间), 计算诱导倍数(诱导倍数=诱导剂组代谢物的生成速率/溶剂对照组的生成速率), 评价阳性诱导剂对CYP1A2、CYP2B6和CYP3A4酶活性的诱导能力。

mRNA表达量分析细胞用预冷的灭菌PBS清洗1次后, 加入RNAiso Plus试剂400 μL, 按说明书提取总RNA, 用微量紫外分光光度计测定RNA的浓度和纯度。以0.75 μg RNA为模板, 按PrimeScript^TM RT reagent kit with gDNA Eraser试剂盒说明书去除基因组DNA后, 加入到30 μL反转录(RT)体系中, RT条件为: 37 ℃ 15 min, 85 ℃ 5 s。使用Premix Ex Taq^TM试剂盒, 按说明书操作进行实时荧光定量PCR。优化后的反应体系为:逆转录产物1.0 μL, PCR上游引物(10 μmol·L^-1) 0.2 μL, PCR下游引物(10 μmol·L^-1) 0.2 μL, 荧光探针溶液(10 μmol·L^-1) 0.4 μL, Premix Ex Taq (Probe qPCR) (2×) 5.0 μL, ROX Reference Dye (50×) 0.2 μL, dH₂O 3.0 μL, 反应条件为: 95 ℃ 30 s; 95 ℃ 5 s, 60 ℃ 30 s, 40个循环。以β-actin为内参基因, 采用2^-ΔΔCt方法分析目的基因在溶剂对照组和诱导剂组表达的相对变化。

分析方法

液质联用方法A (测定人肝微粒体中底物代谢产物)色谱柱为Waters cortess C18⁺ (50×2.1 mm, 2.7 μm), 柱温为38 ℃, 流动相为1 mmol·L^-1甲酸铵(含0.1%甲酸, A)-甲醇(B), 梯度洗脱(0~3.5 min, 10% B; 3.6~8.5 min, 47% B; 8.6~10.5 min, 10% B), 流速为0.6 mL·min^-1, 进样量为4.0 μL。质谱为正离子气动辅助电喷雾离子化(ESI), 离子检测为多重反应监测(multiple reaction monitoring, MRM), 离子化电压为5 kV, 温度为500 ℃, 雾化气、涡轮气和气帘气压力分别为50、50和40 psi, 碰撞气为Medium, 其余参数见表 4。标准含药样本的配制及处理方法为1.5 mL离心管中分别加入相应浓度各CYP450s亚型底物代谢产物工作液10.0 μL和一定蛋白浓度的去活微粒体溶液100 μL, 混匀分别制成含各CYP450s亚型底物代谢物系列浓度的标准含药微粒体(标准曲线范围和质控浓度水平见表 5); 将上述制备好的各CYP450s亚型底物代谢物标准含药微粒体, 分别加入含内标(奥硝唑) 2.00 μmol·L^-1甲醇溶液200 μL, 涡旋3 min, 16 000 r·min^-1离心10 min (4 ℃); 其余同法按“人肝微粒体CYP450s酶活性的评价”项下操作。

液质联用方法B (测定人肝细胞温孵液中底物代谢产物) 除下列条件外, 其他条件同“液质联用方法A”项下内容。流动相为0.1%甲酸(A)-甲醇(B), 梯度洗脱(0~3.0 min, 10% A; 3.1~5.1 min, 48% A; 5.2~7.5 min, 10% A), 进样量为1.0 μL。离子化电压为4 kV, 温度为550 ℃, 雾化气、涡轮气和气帘气压力分别为50、55和35 psi, MRM质谱参数见表 4。标准含药样本的配制及处理方法为取1.5 mL离心管分别加入相应浓度各CYP450s亚型底物代谢产物工作液200 μL和温孵液20.0 μL, 混匀分别制成含各CYP450s亚型底物代谢物系列浓度的标准含药温孵液(标准曲线范围和质控浓度水平见表 4); 将上述制备好的各CYP450s亚型底物代谢物标准温孵液, 分别加入含内标(奥硝唑) 2.00 μmol·L^-1甲醇溶液600 μL, 涡旋3 min, 16 000 r·min^-1离心10 min (4 ℃); 取3种底物代谢物标准温孵液处理后上清液各100 μL于同一离心管中, 涡旋混匀, 进行LC-MS/MS分析。

数据统计 使用GraphPad Prism 5.01软件按照非线性回归计算酶促动力学参数K_m和IC₅₀, 结果以算数均值±标准差($\overline{x}±s$)表示。

结果

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1 分析方法学验证

1.1 特异性

采用液质联用方法A, 在本实验所采用的色谱条件下, 右啡烷、N-去乙基阿莫地喹、1'-羟基咪达唑仑、4'-羟基双氯芬酸、4'-羟基美芬妥英、对乙酰氨基酚、6β-羟基睾酮、羟化安非他酮和内标(奥硝唑)的保留时间见图 1。各待测组分及内标的峰形良好, 无内源性干扰, 方法具有较高的特异性。

1.2 标准曲线及线性范围

分别制备含各CYP450s亚型底物代谢产物的标准曲线, 进行LC-MS/MS分析, 记录色谱图。分别计算样本峰面积A_s和内标峰面积A_i的比ƒ (ƒ = A_s/A_i), 以峰面积比ƒ对浓度作权重回归计算。液质联用方法A和B各CYP450s亚型底物代谢产物的典型回归方程见表 5。

1.3 精密度与准确度

分别制备含最低定量下限、低、中、高4种浓度各CYP450s亚型底物代谢产物的标准含药样本, 共考察3批。进行LC-MS/MS分析, 计算实测浓度、准确度和批内批间精密度。

采用液质联用方法A, 批内和批间精密度的RSD小于15%, 准确度的平均值在91.5%~114.1%内。采用液质联用方法B, 批内和批间精密度的RSD均小于15%, 准确度的平均值在96.6%~106.6%内。

1.4 基质效应

分别按配制低、高2种浓度代谢产物的标准含药样本的量, 分别加入对应各CYP450s亚型底物代谢产物的对照品溶液和内标溶液, 涡旋混匀, 于35 ℃水浴中以氮气流吹干。残渣分别用流动相和空白微粒体提取液复溶后, 进行LC-MS/MS分析, 计算各CYP450s亚型底物代谢产物的基质效应^[6]。

液质联用方法A, 基质效应的平均值在94.3%~106.8%内。液质联用方法B, 基质效应的平均值在93.5%~107.9%内。

1.5 提取回收率

分别制备含低、中、高3种浓度各CYP450s亚型底物代谢产物的标准含药样本; 同时按配制低、中、高3种浓度各CYP450s亚型底物代谢产物的标准含药样本的量分别加入对应代谢产物的对照品溶液和内标, 涡旋混匀, 于35 ℃水浴中以氮气流吹干。残渣用空白样本提取液复溶后, 进行LC-MS/MS分析。分别计算低、中、高3种浓度的提取回收率。

液质联用方法A, 底物代谢产物及内标提取回收率的平均值为93.6%~112.0%。液质联用方法B:底物代谢产物及内标提取回收率的平均值为80.9%~98.7%。

1.6 稳定性

分别考察低、中、高3种浓度的各CYP450s亚型底物代谢产物的标准含药样本在实验过程中的稳定性。液质联用方法A, 标准含药微粒体样本于室温放置22 h、-40 ℃冰冻放置41天及待测溶液在自动进样器(8 ℃)中放置24 h后, 各CYP450s亚型底物代谢产物均稳定。液质联用方法B, 标准含药温孵液样本, 于室温放置20 h, -40 ℃冰冻放置34天和待测溶液在进样器(8 ℃)中放置22 h, 各CYP450s亚型底物代谢产物均稳定。

1.7 残留效应

分别将含各CYP450s亚型底物代谢产物浓度为标准曲线最高点的标准含药样本和空白样本交替进样, 进行LC-MS/MS分析, 记录色谱图, 分别记录各CYP450s亚型底物代谢物的面积As和内标奥硝唑的峰面积Ai。液质联用方法A和B均无残留。

2 人肝微粒体酶活性和阳性对照IC₅₀的测定结果

2.1 人肝微粒体酶活性测定

本实验条件下, 采用探针底物法测定人肝微粒体各亚型CYP1A2、CYP2B6、CYP2C8、CYP2C9、CYP2C19、CYP2D6和CYP3A4/5的酶活性数据见表 6。所得人肝微粒体各亚型的酶活性数据基本在FDA^{[8, 9]}推荐结果的范围内, 表明所建立的微粒体温孵体系良好, 可用于后续研究。

2.2 人肝微粒体阳性对照IC₅₀测定结果

本实验条件下, 阳性对照α-萘黄酮、塞替派、槲皮素、磺胺苯吡唑、噻氯匹定、奎尼丁和酮康唑对相应CYP450酶各亚型均表现出明显的抑制作用(IC₅₀均小于10.0 μmol·L^-1, 表 6), 各亚型阳性对照的抑制曲线见图 2。表明本实验建立的微粒体温孵体系可用于抑制作用的评价。

3 人原代肝细胞CYP酶活性和mRNA表达量的测定

3.1 人原代肝细胞CYP酶活性

与溶剂对照组相比, LTG、ONO和YOW等3个批号肝细胞经阳性诱导剂奥美拉唑处理后, CYP1A2活性的诱导倍数分别为33.9、28.7和52.8倍; 经阳性诱导剂苯巴比妥处理后, CYP2B6活性的诱导倍数分别为8.33、7.85和6.16;经阳性诱导剂利福平处理后, CYP3A4活性的诱导倍数分别为6.63、46.8和11.0倍。结果表明, 各批次人原代肝细胞各亚型CYP450s活性均被诱导, 本实验建立的人肝细胞温孵体系可用于诱导作用的评价。

3.2 人原代肝细胞mRNA表达量

与溶剂对照组相比, LTG、ONO和YOW等3个批号肝细胞经阳性诱导剂奥美拉唑处理后, CYP1A2 mRNA表达的诱导倍数分别为77.4、28.0和50.5倍; 经阳性诱导剂苯巴比妥处理后, CYP2B6 mRNA表达的诱导倍数分别为23.7、5.57和5.30倍; 经阳性诱导剂利福平处理后, CYP3A4 mRNA表达的诱导倍数分别为29.8、165和22.2倍。结果表明, 各批次人原代肝细胞各亚型CYP450s表达均被诱导, 本实验建立的温孵体系可用于人肝细胞诱导作用的评价。

讨论

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本实验将CYP450酶各亚型的探针底物分别与肝微粒体温孵, 排除了混和探针底物与微粒体同时温孵对实验结果可能造成的影响; 将经沉淀剂处理后的单个底物温孵样本合并, 应用LC-MS/MS同时测定各底物的代谢产物, 提高了分析的通量。为了准确表示酶的活性, 必须保证实验时采用酶促反应开始时的初速度, 即要优化反应条件, 使代谢物的形成速率与温孵时间及蛋白含量成线性^{[6, 8, 9]}。实验在采用酶促反应开始的初速度及兼顾分析方法灵敏度的前提下, 尽量选择低的蛋白浓度, 避免因底物或抑制剂与蛋白产生非特异性的结合, 导致结果偏差^{[6, 9]}。IC₅₀抑制实验在测定或已知该批次微粒体各底物K_m值后进行, 通常在兼顾分析方法灵敏度的前提下, 选择小于各底物K_m值作为抑制实验的底物浓度^{[6, 9]}。如果待测药物能对CYP450酶产生抑制作用, 则应用合适的酶促动力学模型可以测得IC₅₀。测定IC₅₀值可快速初步了解药物对代谢酶是否有强抑制作用, 若IC₅₀值 < 1.00 μmol·L^-1, 则初步表明药物对该酶的抑制能力强; 若1.00 μmol·L^-1 < IC₅₀值 < 10.0 μmol·L^-1, 则表明药物对该酶的抑制能力中等; 若IC₅₀值 > 30.0 μmol·L^-1, 则表明药物对该酶的抑制作用较弱。

国家食品药品监督管理总局的《药物相互作用研究指导原则》 (2012年版)中指出CYP2D6酶未显示有可诱导性, CYP2C、CYP2B和ABCB1 (P-gp)转运体与CYP3A具有协同诱导作用, CYP3A似乎对所有已知协同诱导物均敏感。因此, 为了评价研究药物是否对CYP1A2、CYP2C8、CYP2C9、CYP2C19、或CYP3A有诱导作用, 最初体外诱导评价可能仅包括CYP1A2和CYP3A。如果体外研究结果表明, 研究药物对CYP3A代谢不具有诱导作用, 那么就不需要在这些酶诱导水平下进行研究药物与经CYP2C/ CYP2B及CYP3A消除的合用药物的体内相互作用研究。CYP2B6介导的药物相互作用是重要的相互作用, 需进行基于该酶的相互作用的体外研究。其他CYP酶(包括CYP2A6和CYP2E1)被认为较少参与具有临床重要性的药物相互作用。故本研究选择对人原代肝细胞CYP1A2、CYP2B6和CYP3A进行体外诱导研究。

本实验分别建立的测定人肝微粒体及人原代肝细胞温孵液中各CYP450酶亚型底物代谢产物的LC-MS/MS法, 验证结果显示分析方法符合生物样本分析的要求。测定的人肝微粒体CYP450酶7种主要亚型的酶活性数据基本在FDA推荐结果的范围内, 分别应用已知抑制剂对抑制实验和诱导实验体系进行验证的结果表明温孵体系可用于药物对CYP450诱导与抑制作用的评价。本研究建立的药物对CYP450诱导与抑制作用的体外实验方法简便、可靠, 可用于新药候选化合物酶诱导或抑制性质的早期评价及临床合用药物代谢性相互作用的体外评价。

基金

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国家自然科学基金资助项目(81503160)
国家自然科学基金资助项目(81673515)
江苏省自然科学基金资助项目(BK20161591)
江苏省“六大人才高峰”项目(2014-YY-001)
江苏省医学重点人才基金项目(H201108)
药物质量与安全预警教育部重点实验室资助项目(DQCP2015MS07)

参考文献

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文献

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2017年第52卷第12期

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文章信息

doi: 10.16438/j.0513-4870.2017-0700

接收时间：2017-07-18
首发时间：2026-01-14
出版时间：2017-12-12

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收稿日期：2017-07-18
修回日期：2017-08-29

基金

国家自然科学基金资助项目(81503160)

国家自然科学基金资助项目(81673515)

江苏省自然科学基金资助项目(BK20161591)

江苏省“六大人才高峰”项目(2014-YY-001)

江苏省医学重点人才基金项目(H201108)

药物质量与安全预警教育部重点实验室资助项目(DQCP2015MS07)

作者信息

1.南京医科大学第一附属医院, 江苏南京 210029

2.中国药科大学药物质量与安全预警教育部重点实验室, 江苏南京 210009

3.中国药科大学药物分析系, 江苏南京 210009

4.南京科利泰医药科技有限公司, 江苏南京 211100

通讯作者:

* 丁黎, Tel:86-25-83271289, E-mail:dinglihg@sina.com;

王永庆, Tel:86-25-68136984, E-mail:wyqjsph@163.com

参考文献

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https://castjournals.cast.org.cn/joweb/yxxb/CN/10.16438/j.0513-4870.2017-0700

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Target	mRNA sequence	Primer/probe	Sequence
CYP1A2	NM_000761.3	5'-Primer	5'-AAA TGC TGT GTC TTC GTA AAC CAG-3'
		3'-Primer	5'-CGC TCA GGC CGG AAC TC-3'
		Probe	5'-(FAM) cca gag ctg tgg gag gac ccc tc (Tamra)-3'
CYP2B6	NM_000767.4	5'-Primer	5'-GCC ACC CTA ACA CCC ATG AC-3'
		3'-Primer	5'-TGA GTA GGC CTC TTC TAT CCA TCT G-3'
		Probe	5'-(FAM) cac cag ggc ccc gcc ctc t (Tamra)-3'
CYP3A4	NM_017460.5	5'-Primer	5'-GTG TGT TTC CAA GAG AAG TTA CAA ATT T-3'
		3'-Primer	5'-CCA CTC GGT GCT TTT GTG TAT C-3'
		Probe	5'-(FAM) cga ggc gac ttt ctt tca tcc ttt tta cag a (Tamra)-3'
β-Actin	NM_001101	5'-Primer	5'-GAC TAC CTC ATG AAG ATC CTC ACC-3'
		3'-Primer	5'-TCT CCT TAA TGT CAC GCA CGA TT-3'
		Probe	5'-(FAM) CGG CTA CAG CTT CAC CAC CAC GGC (TAMRA)-3'

Target

mRNA sequence

Primer/probe

Sequence

CYP1A2

NM_000761.3

5'-Primer

5'-AAA TGC TGT GTC TTC GTA AAC CAG-3'

3'-Primer

5'-CGC TCA GGC CGG AAC TC-3'

Probe

5'-(FAM) cca gag ctg tgg gag gac ccc tc (Tamra)-3'

CYP2B6

NM_000767.4

5'-Primer

5'-GCC ACC CTA ACA CCC ATG AC-3'

3'-Primer

5'-TGA GTA GGC CTC TTC TAT CCA TCT G-3'

Probe

5'-(FAM) cac cag ggc ccc gcc ctc t (Tamra)-3'

CYP3A4

NM_017460.5

5'-Primer

5'-GTG TGT TTC CAA GAG AAG TTA CAA ATT T-3'

3'-Primer

5'-CCA CTC GGT GCT TTT GTG TAT C-3'

Probe

5'-(FAM) cga ggc gac ttt ctt tca tcc ttt tta cag a (Tamra)-3'

β-Actin

NM_001101

5'-Primer

5'-GAC TAC CTC ATG AAG ATC CTC ACC-3'

3'-Primer

5'-TCT CCT TAA TGT CAC GCA CGA TT-3'

Probe

5'-(FAM) CGG CTA CAG CTT CAC CAC CAC GGC (TAMRA)-3'

CYP450 enzyme	Substrate	Incubation condition
CYP1A2	Phenacetin	0.250	10	5.00, 10.0, 30.0, 50.0, 100, 200, 400
CYP2B6	Bupropion	0.100	15	5.00, 10.0, 30.0, 50.0, 100, 200, 500
CYP2C8	Amodiaquine	0.100	10	0.300, 0.600, 1.00, 2.00, 5.00, 10.0, 20.0
CYP2C9	Diclofenac	0.250	10	1.00, 2.00, 5.00, 10.0, 50.0, 100, 200
CYP2C19	S-Mephenytoin	1.00	60	2.00, 5.00, 10.0, 30.0, 60.0, 100, 200
CYP2D6	Dextromethorphan	0.100	20	0.100, 0.500, 1.00, 5.00, 10.0, 20.0, 30.0
CYP3A4/5	Midazolam	0.100	5	0.200, 0.500, 1.00, 3.00, 10.0, 20.0, 50.0
CYP3A4/5	Testosterone	0.100	15	5.00, 10.0, 20.0, 50.0, 100.0, 200, 300

CYP450 enzyme

Substrate

Incubation condition

Enzyme activity

Protein concentration/mg·mL^-1

Time/min

Substrate concentration/μmol·L^-1

CYP1A2

Phenacetin

0.250

5.00, 10.0, 30.0, 50.0, 100, 200, 400

CYP2B6

Bupropion

0.100

5.00, 10.0, 30.0, 50.0, 100, 200, 500

CYP2C8

Amodiaquine

0.100

0.300, 0.600, 1.00, 2.00, 5.00, 10.0, 20.0

CYP2C9

Diclofenac

0.250

1.00, 2.00, 5.00, 10.0, 50.0, 100, 200

CYP2C19

S-Mephenytoin

1.00

2.00, 5.00, 10.0, 30.0, 60.0, 100, 200

CYP2D6

Dextromethorphan

0.100

0.100, 0.500, 1.00, 5.00, 10.0, 20.0, 30.0

CYP3A4/5

Midazolam

0.100

0.200, 0.500, 1.00, 3.00, 10.0, 20.0, 50.0

CYP3A4/5

Testosterone

0.100

5.00, 10.0, 20.0, 50.0, 100.0, 200, 300

CYP450 enzyme	Substrate	Substrate concentration /μmol·L^-1	Positive control of inhibitor	Positive control concentration/μmol·L^-1
CYP1A2	Phenacetin	150	α-Naphthoflavone	0, 0.00100, 0.00500, 0.0100, 0.0300, 0.0600, 0.100, 1.00, 10.0
CYP2B6	Bupropion	100	Thiotepa	0, 0.100, 0.500, 1.00, 5.00, 10.0, 20.0, 50.0, 100
CYP2C8	Amodiaquine	2.00	Quercetin	0, 0.0500, 0.100, 0.500, 1.00, 2.00, 5.00, 10.0, 20.0
CYP2C9	Diclofenac	10.0	Sulfaphenazole	0, 0.0100, 0.0500, 0.100, 0.300, 0.600, 1.00, 3.00, 10.0
CYP2C19	S-Mephenytoin	35.0	Ticlopidine	0, 0.00500, 0.0500, 0.500, 2.00, 5.00, 10.0, 20.0, 50.0
CYP2D6	Dextromethorphan	10.0	Quinidine	0, 0.100, 0.200, 0.500, 1.00, 2.00, 5.00, 10.0, 20.0
CYP3A4/5	Midazolam	2.00	Ketoconazole	0, 0.00100, 0.0100, 0.0500, 0.100, 0.500, 1.00, 5.00, 10.0
CYP3A4/5	Testosterone	50.0	Ketoconazole	0, 0.000100, 0.000500, 0.00100, 0.00500, 0.0100, 0.100, 1.00, 5.00

CYP450 enzyme

Substrate

Substrate concentration /μmol·L^-1

Positive control of inhibitor

Positive control concentration/μmol·L^-1

CYP1A2

Phenacetin

150

α-Naphthoflavone

0, 0.00100, 0.00500, 0.0100, 0.0300, 0.0600, 0.100, 1.00, 10.0

CYP2B6

Bupropion

100

Thiotepa

0, 0.100, 0.500, 1.00, 5.00, 10.0, 20.0, 50.0, 100

CYP2C8

Amodiaquine

2.00

Quercetin

0, 0.0500, 0.100, 0.500, 1.00, 2.00, 5.00, 10.0, 20.0

CYP2C9

Diclofenac

10.0

Sulfaphenazole

0, 0.0100, 0.0500, 0.100, 0.300, 0.600, 1.00, 3.00, 10.0

CYP2C19

S-Mephenytoin

35.0

Ticlopidine

0, 0.00500, 0.0500, 0.500, 2.00, 5.00, 10.0, 20.0, 50.0

CYP2D6

Dextromethorphan

10.0

Quinidine

0, 0.100, 0.200, 0.500, 1.00, 2.00, 5.00, 10.0, 20.0

CYP3A4/5

Midazolam

2.00

Ketoconazole

0, 0.00100, 0.0100, 0.0500, 0.100, 0.500, 1.00, 5.00, 10.0

CYP3A4/5

Testosterone

50.0

Ketoconazole

0, 0.000100, 0.000500, 0.00100, 0.00500, 0.0100, 0.100, 1.00, 5.00

Study	CYP450 enzyme	Analyte	MRM transition	DP/V	EP/V	CE/V	CXP/V
Inhibition	CYP1A2	Acetaminophen	152.2 → 110.1	100	10	21	12
	CYP2B6	Hydroxybupropion	256.1 → 238.1	65	7	20	18
	CYP2C8	N-Desethyl amodiaquine	328.2 → 283.2	120	10	26	22
	CYP2C9	4'-Hydroxydiclofenac	312.1 → 266.1	100	10	17.5	20
	CYP2C19	4'-Hydroxymephenytoin	235.2 → 150.1	120	10	25	20
	CYP2D6	Dextrorphan	258.3 → 157.1	130	10	47	13
	CYP3A4/5	1'-Hydroxymidazolam	305.3 → 269.3	120	12	18	18
	CYP3A4/5	6β-Hydroxytestosterone	342.1 → 324.1	120	10	28.5	22
	IS	Ornidazole	220.1 → 128.0	120	10	20	13
Induction	CYP1A2	Acetaminophen	152.2 → 110.1	120	10	21	12
	CYP2B6	Hydroxybupropion	256.1 → 238.1	120	10	20	18
	CYP3A4/5	6β-Hydroxytestosterone	305.1 → 324.1	120	10	18	18
	IS	Ornidazole	220.1 → 128.0	120	10	20	13

Study

CYP450 enzyme

Analyte

MRM transition

DP/V

EP/V

CE/V

CXP/V

Inhibition

CYP1A2

Acetaminophen

152.2 → 110.1

100

CYP2B6

Hydroxybupropion

256.1 → 238.1

CYP2C8

N-Desethyl amodiaquine

328.2 → 283.2

120

CYP2C9

4'-Hydroxydiclofenac

312.1 → 266.1

100

17.5

CYP2C19

4'-Hydroxymephenytoin

235.2 → 150.1

120

CYP2D6

Dextrorphan

258.3 → 157.1

130

CYP3A4/5

1'-Hydroxymidazolam

305.3 → 269.3

120

CYP3A4/5

6β-Hydroxytestosterone

342.1 → 324.1

120

28.5

Ornidazole

220.1 → 128.0

120

Induction

CYP1A2

Acetaminophen

152.2 → 110.1

120

CYP2B6

Hydroxybupropion

256.1 → 238.1

120

CYP3A4/5

6β-Hydroxytestosterone

305.1 → 324.1

120

Ornidazole

220.1 → 128.0

120

Study	Analyte	SC range/nmol·L^-1	QC level/nmol·L^-1	Typical standard curve
Inhibition	Acetaminophen	25.0-2 500	60.0, 300, 2 000	ƒ = 4.65e^-5 × C -1.18e^-4, r = 0.999 0
	Hydroxybupropion	10.0-1 000	24.0, 120, 800	ƒ = 1.75e^-4 × C -2.22e^-4, r = 0.999 7
	N-Desethyl amodiaquine	5.00-500	12.0, 60.0, 400	ƒ = 2.90e^-4 × C -9.77e^-5, r = 0.999 9
	4'-Hydroxydiclofenac	250-25 000	600, 3 000, 20 000	ƒ = 5.04e^-5 × C + 2.16e^-4, r = 0.999 9
	4'-Hydroxymephenytoin	100-10 000	240, 1 200, 8 000	ƒ = 2.16e^-5 × C -1.75e^-5, r = 0.999 7
	Dextrorphan	5.00-500	12.0, 60.0, 400	ƒ = 1.34e^-4 × C -9.90e^-5, r = 0.999 9
	1'-Hydroxymidazolam	5.00-500	12.0, 60.0, 400	ƒ = 4.19e^-4× C -1.99e^-4, r = 0.999 3
	6β-Hydroxytestosterone	100-10 000	240, 1 200, 8 000	ƒ = 2.72e^-5 × C -5.05e^-5, r = 0.999 8
Induction	Acetaminophen	100-40 000	250, 3 000, 32 000	ƒ = 6.13e^-2 × C + -5.41e^-4, r = 0.999 2
	Hydroxybupropion	100-40 000	250, 3 000, 32 000	ƒ = 2.41e^-1 × C + 1.25 e^-3, r = 0.997 6
	6β-Hydroxytestosterone	100-40 000	250, 3 000, 32 000	ƒ = 3.61e^-2 × C -3.67e^-4, r = 0.999 0

Study

Analyte

SC range/nmol·L^-1

QC level/nmol·L^-1

Typical standard curve

Inhibition

Acetaminophen

25.0-2 500

60.0, 300, 2 000

ƒ = 4.65e^-5 × C -1.18e^-4, r = 0.999 0

Hydroxybupropion

10.0-1 000

24.0, 120, 800

ƒ = 1.75e^-4 × C -2.22e^-4, r = 0.999 7

N-Desethyl amodiaquine

5.00-500

12.0, 60.0, 400

ƒ = 2.90e^-4 × C -9.77e^-5, r = 0.999 9

4'-Hydroxydiclofenac

250-25 000

600, 3 000, 20 000

ƒ = 5.04e^-5 × C + 2.16e^-4, r = 0.999 9

4'-Hydroxymephenytoin

100-10 000

240, 1 200, 8 000

ƒ = 2.16e^-5 × C -1.75e^-5, r = 0.999 7

Dextrorphan

5.00-500

12.0, 60.0, 400

ƒ = 1.34e^-4 × C -9.90e^-5, r = 0.999 9

1'-Hydroxymidazolam

5.00-500

12.0, 60.0, 400

ƒ = 4.19e^-4× C -1.99e^-4, r = 0.999 3

6β-Hydroxytestosterone

100-10 000

240, 1 200, 8 000

ƒ = 2.72e^-5 × C -5.05e^-5, r = 0.999 8

Induction

Acetaminophen

100-40 000

250, 3 000, 32 000

ƒ = 6.13e^-2 × C + -5.41e^-4, r = 0.999 2

Hydroxybupropion

100-40 000

250, 3 000, 32 000

ƒ = 2.41e^-1 × C + 1.25 e^-3, r = 0.997 6

6β-Hydroxytestosterone

100-40 000

250, 3 000, 32 000

ƒ = 3.61e^-2 × C -3.67e^-4, r = 0.999 0

CYP450 enzyme	Substrate	Positive control	K_m/μmol·L^-1	IC₅₀/μmol·L^-1
CYP1A2	Phenacetin	α-Naphthoflavone	114 ± 33	1.7-152	0.022 0 ± 0.012 3
CYP2B6	Bupropion	Thiotepa	158 ± 108	67-168	0.444 ± 0.068
CYP2C8	Amodiaquine	Quercetin	2.61 ± 0.06	2.4	1.55 ± 0.69
CYP2C9	Diclofenac	Sulfaphenazole	9.16 ± 1.17	3.4-52	0.966 ± 0.348
CYP2C19	S-Mephenytoin	Ticlopidine	16.6 ± 6.6	13-35	0.223 ± 0.106
CYP2D6	Dextromethorphan	Quinidine	20.2 ± 12.8	0.44-8.5	0.685 ± 0.266
CYP3A4/5	Midazolam	Ketoconazole	1.60 ± 0.34	1.0-14	0.773 ± 0.178
CYP3A4/5	Testosterone	Ketoconazole	78.1 ± 4.7	52-94	0.968 ± 0.144

CYP450 enzyme

Substrate

Positive control

K_m/μmol·L^-1

IC₅₀/μmol·L^-1

Result

FDA^{[8, 9]}

CYP1A2

Phenacetin

α-Naphthoflavone

114 ± 33

1.7-152

0.022 0 ± 0.012 3

CYP2B6

Bupropion

Thiotepa

158 ± 108

67-168

0.444 ± 0.068

CYP2C8

Amodiaquine

Quercetin

2.61 ± 0.06

2.4

1.55 ± 0.69

CYP2C9

Diclofenac

Sulfaphenazole

9.16 ± 1.17

3.4-52

0.966 ± 0.348

CYP2C19

S-Mephenytoin

Ticlopidine

16.6 ± 6.6

13-35

0.223 ± 0.106

CYP2D6

Dextromethorphan

Quinidine

20.2 ± 12.8

0.44-8.5

0.685 ± 0.266

CYP3A4/5

Midazolam

Ketoconazole

1.60 ± 0.34

1.0-14

0.773 ± 0.178

CYP3A4/5

Testosterone

Ketoconazole

78.1 ± 4.7

52-94

0.968 ± 0.144