A radiometric assay for debrisoquine 4-hydroxylase in human liver microsomes

1984 ◽  
Vol 62 (1) ◽  
pp. 84-88 ◽  
Author(s):  
M. Nakano ◽  
T. Inaba
Keyword(s):  
Human Liver ◽  
High Performance ◽  
Liquid Scintillation ◽  
Liver Microsomes ◽  
Microsomal Protein ◽  
Scintillation Counting ◽  
Human Liver Microsomes ◽  
Hydroxylase Activity ◽  
Radiometric Assay ◽  
Layer Chromatography

A radiometric method to assay debrisoquine 4-hydroxylase activity in human liver microsomes was established. Following incubation with 14C-labelled debrisoquine, unreacted debrisoquine was extracted with chloroform; 4-hydroxydcbrisoquine was derivatized with hexafluoroacetylacetone, extracted, and subjected to high performance thin-layer chromatography (HP-TLC) followed by liquid scintillation counting. The Km values for debrisoquine 4-hydroxylase were 70 and 120 μM and the Vmax values were 8 and 24 pmol per milligram microsomal protein per minute. By application of this assay, it was possible to show that 4-hydroxydebrisoquine formation was competitively inhibited by sparteine. Antipyrine up to a concentration of 4 mM had no effect.

Inhibition of Coumarin 7-Hydroxylase Activity in Human Liver Microsomes

1997 ◽  
Vol 341 (1) ◽  
pp. 47-61 ◽  
Author(s):  
Alison J. Draper ◽  
Ajay Madan ◽  
Andrew Parkinson
Keyword(s):  
Human Liver ◽  
Liver Microsomes ◽  
Human Liver Microsomes ◽  
Hydroxylase Activity ◽  
Coumarin 7

scholarly journals The Potential for CYP2D6 Inhibition Screening Using a Novel Scintillation Proximity Assay-Based Approach

2001 ◽  
Vol 6 (4) ◽  
pp. 225-231 ◽  
Author(s):  
Enock Delaporte ◽  
Donald E. Slaughter ◽  
Marjorie A. Egan ◽  
Gregory J. Gatto ◽  
Albie Santos ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
High Throughput ◽  
Human Liver ◽  
High Performance ◽  
Liver Microsomes ◽  
Inhibitory Effect ◽  
Human Liver Microsomes ◽  
Kinetics Parameters ◽  
Scintillation Proximity Assay ◽  
Demethylase Activity

High throughput inhibition screens for human cytochrome P450s (CYPs) are being used in preclinical drug metabolism to support drug discovery programs. The versatility of scintillation proximity assay (SPA) technology has enabled the development of a homogeneous high throughput assay for cytochrome P450 2D6 (CYP2D6) inhibition screen using [O-methyl-'4C]dextromethorphan as substrate. The basis of the assay was the trapping of the 0- demethylation product, [14C]HCHO, on SPA beads. Enzyme kinetics parameters Vm,,. and apparent Ki,, determined using pooled human liver microsomes and microsomes from baculovirus cells coexpressing human CYP2D6 and NADPH-cytochrome P450 reductase, were 245 pmol [14C]HCHO/min/mg protein and 11,tM, and 27 pmol ['4C]HCHO/min/pmol and 1.6,uM, respectively. In incubations containing either pooled microsomes or recombinant CYP2D6, [14C]dextromethorphan 0-demethylase activity was inhibited in the presence of quinidine (IC50 = 1.0,uM and 20 nM, respectively). By comparison, inhibitors selective for other CYP isoforms were relatively weak (IC50 > 25 tM). In agreement, a selective CYP2D6 inhibitory monoclonal antibody caused greater than 90% inhibition of [14C]dextromethorphan O-demethylase activity in human liver microsomes, whereas CYP2C9/19- and CYP3A4/5-selective antibodies elicited a minimal inhibitory effect. SPA-based [14C]dextromethorphan 0-demethylase activity was also shown to correlate (r2 = 0.6) with dextromethorphan O-demethylase measured by high-performance liquid chromatography in a bank of human liver microsomes (N = 15 different organ donors). In a series of known CYP2D6 inhibitors/substrates, the SPA-based assay resolved potent inhibitors (IC50 <2 μM) from weak inhibitors (IC50 ≥ 20 μM). It is concluded that the SPA-based assay described herein is suitable for CYP2D6 inhibition screening using either native human liver microsomes or cDNA-expressed CYP2D6.

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