Validation of cytochrome P450 time-dependent inhibition assays: a two-time point IC50 shift approach facilitates kinact assay design.
1. Recent guidance from the US Food and Drug Administration (USFDA) has advocated testing of time-dependent inhibition of cytochrome P450 (CYP), which can be addressed by performing IC(50) shift as well as K(I)/k(inact) determinations. 2. Direct (IC(50), K(i)) and time-dependent inhibition (IC(50) shift, K(I)/k(inact)) assays were validated in human liver microsomes with liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis for the following enzyme/substrate/inhibitor combinations: CYP1A2/phenacetin/alpha-naphthoflavone/furafylline, CYP2C8/amodiaquine/montelukast/gemfibrozil-1-O-beta-glucuronide, CYP2C9/diclofenac/sulfaphenazole/tienilic acid, CYP2C19/S-mephenytoin/S-benzylnirvanol/S-fluoxetine, CYP2D6/dextromethorphan/quinidine/paroxetine, and CYP3A4/midazolam/testosterone/ketoconazole/azamulin/verapamil/diltiazem. IC(50) shift assays were performed with two pre-incubation time points (10 and 30 min) to facilitate k(inact) assay design. 3. Data obtained show good agreement with literature values. For rapid acting inhibitors, such as azamulin/CYP3A4 and tienilic acid/CYP2C9, the IC(50) shifts were similar at both time points suggesting a short maximum pre-incubation time with closely spaced time points for the K(I)/k(inact) assay. Slow acting inhibitors (such as verapamil/CYP3A4 or S-fluoxetine/CYP2C19) showed an increase in IC(50) shift between 10 and 30 min suggesting a longer maximum pre-incubation time with wider spacing of time points for K(I)/k(inact). 4. The two-time point IC(50) shift experiment proved to be an excellent method for the selection of appropriate K(I)/k(inact) assay parameters and is suitable for the routine analysis of P450 inhibition by drug candidates.
Perloff
E S
ES
BD Biosciences Discovery Labware, BD Gentest Contract Research Services, Woburn, MA 01801, USA. elke_perloff@bd.com
Mason
A K
AK
Dehal
S S
SS
Blanchard
A P
AP
Morgan
L
L
Ho
T
T
Dandeneau
A
A
Crocker
R M
RM
Chandler
C M
CM
Boily
N
N
Crespi
C L
CL
Stresser
D M
DM
D002853:Chromatography, Liquid; D065607:Cytochrome P-450 Enzyme Inhibitors; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology* / Q000633:toxicity; D006801:Humans; D020128:Inhibitory Concentration 50; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D013997:Time Factors
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors