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A fully automated assay to determine the enzymology of drug oxidation by …


Biology Articles » Biochemistry » Enzymology » Automated Definition of the Enzymology of Drug Oxidation by the Major Human Drug Metabolizing Cytochrome P450s » Tables

Tables
- Automated Definition of the Enzymology of Drug Oxidation by the Major Human Drug Metabolizing Cytochrome P450s




TABLE 1
CYP isoform characterization of individual HLM pools

 


Isoform Probe Batch 1 Batch 2 Batch 3



pmol/min/mg
P450 content CO difference spectrum 320a 280a 430a
P450 reductase Cytochrome c reduction 69000 84000 75000
CYP 7-Ethoxycoumarin O-demethylation 255 210 240
CYP1A2 Phenacetin O-dethylation 366 310 193
CYP2A6 Coumarin 7-hydroxylation 17200 81900 58600
CYP2E1 Chlorzoxazone 6-hydroxyline 1010 1911 1441
CYP2C Mephenytoin 4-hydroxylation 27 233 11
CYP2D6 Dextromethorphan O-demethylation 200 84 106
CYP3A Testosterone 6beta-hydroxylation 2600 1500 1900
CYP4A Lauric acid 11-hydroxylation 1000 1600 1600

a  Picomoles of CYP per milligram of microsomal protein.


 

 



TABLE 2
Estimated levels of the five major isoforms in HLM pools

Mean total level = 320 pmol of CYP · mg-1 of microsomal protein.


Isoform Mean References


%
CYP1A2 13 Shimada et al. (1994), Guengerich and Turvy (1991), Belloc et al. (1996)
CYP2C9 20 Shimada et al. (1994), Becquemont et al. (1998), Wester et al. (2000), Inoue et al. (1997), Lasker et al. (1998)
CYP2C19 4 Wester et al. (2000), Inoue et al. (1997), Lasker et al. (1998)
CYP2D6 2 Shimada et al. (1994), Becquemont et al. (1998), Imaoka et al. (1996)
CYP3A4 30 Shimada et al. (1994), Belloc et al. (1996), Becquemont et al. (1998)


 

 



TABLE 3
CLint of marker substrates in different HLM pools and literature values

 


Compound CLint
References
Batch 1 Batch 2 Batch 3 Mean Literature


µl · min-1mg-1
Tolbutamide


0.7a 0.5-2.5; 1 ± 0.2 Iwatsubo et al. (1997), Doecke et al. (1991), Back et al. (1988), Bourrie et al. (1996), Obach (1999)
Diazepam 3  ± 1 2 2 3  ± 1 2.6  ± 0.8 Obach (1999)
Metoprolol 7  ± 1 5 5 6  ± 1 14, 19 Mautz et al. (1995)
Ibuprofen 14 5 5 8  ± 2 9.6  ± 1.1 Obach (1999)
Propranolol 16  ± 3 16 15 15  ± 0 11-60 Lave et al. (1997), Obach (1997), Otton et al. (1990)
Dextromethorphan 29  ± 3 30 33 29  ± 3  25-103 Kerry et al. (1994), Broly et al. (1990)
Omeprazole 43  ± 7 11 15 34  ± 14 18, 26 Andersson et al. (1993), Kobayashi et al. (1994)
Diltiazem 56  ± 7 22 22 45  ± 16 17  ± 2 Obach (1999)
Testosterone 81  ± 12 23 35 60  ± 18
Verapamil 380  ± 44 111 114 256  ± 100 139  ± 3 Obach (1999)

a  Determined from Vmax/Km. Where determined, results are presented as mean ± S.D.


 

 



TABLE 4
Determination of CYP Clint of individual human CYPs to oxidative metabolism for marker substrates

 


Compound CLint
CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4


µl · min-1pmol-1
Tolbutamide N.D.a 0.014b 0.03  ± 0.01 N.D. N.D.
Diazepam N.D. N.D. 0.17  ± 0.05 N.D. N.D.
Metoprolol N.D. N.D. N.D. 1.03  ± 0.07 N.D.
Ibuprofen N.D. 0.29 ± 0.09 0.17  ± 0.01 N.D. N.D.
Propranolol 0.34 ± 0.03 N.D. 0.66  ± 0.06 5.07  ± 2.80 N.D.
Dextromethorphan N.D. N.D. 0.16  ± 0.10 1.97  ± 0.24 N.D.
Omeprazole N.D. N.D. 6.95  ± 1.93 N.D. 0.44  ± 0.06
Diltiazem N.D. N.D. 0.30  ± 0.04 N.D. 0.58  ± 0.04
Testosterone N.D. N.D. N.D. N.D. 0.42  ± 0.03
Verapamil N.D. N.D. N.D. N.D. 1.19  ± 0.12

a  N.D., not detectable (<0.02 µl · min-1 · pmol-1).
b  Determined from Vmax/Km. Mean ± S.D. for n = 3 separate experiments.


 

 



TABLE 5
Mean percentage contribution of individual CYPs to oxidative metabolism

 


Compound CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4 Literature


%
Tolbutamide
70 30

2C
Diazepam

100

2C19>3A
Metoprolol


100
2D6
Ibuprofen
90 10

2C9
Propranolol 26
15 59
2D6, 1A2, 2C19
Dextromethorphan

14 86
2D6>3A
Omeprazole

68
32 2C19z.Gt.gif3A
Diltiazem

7
93 3A
Testosterone



100 3A
Verapamil



100 3A


 

 



TABLE 6
Determination of CYP Clint for marker and AR-C compounds

 


Compound CLint
HLM CYP


µl · min-1mg-1
Tolbutamide 0.7a 1.3a
Diazepam 3  ± 1 2  ± 1
Metoprolol 6  ± 1 7  ± 0
Ibuprofen 8  ± 2 20  ± 5
Propranolol 15  ± 0 55  ± 15
Dextromethorphan 29  ± 3 14  ± 1
Omeprazole 34  ± 14 131  ± 25
Diltiazem 45  ± 16 60  ± 3
Testosterone 60  ± 18 40  ± 3
Verapamil 256  ± 100 114  ± 11

a  Determined from Vmax/Km. Mean ± S.D.



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