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Differentiation of Reversible and MechanismBased Inhibitors of Human CYP MediatedReactions by an IC50 Shift Approach
Zhiming Wen, Joseph Rager, Praveen Srivastava, Carol Kissa, Nicholas Oranzi, Jibin Li, Sid Bhoopathy, Albert Owen, and Chris Bode
Absorption Systems, Exton, PA 19341, USA
25
-1
0
1
20
-1
1
Phenacetin
150
Acetaminophen
+ESI, 152/110
2A6
Coumarin
6
7-OH Coumarin
-ESI, 161/133
2B6
Bupropion
100
Hydroxybupropion
+ESI, 256/238
2C9
Diclofenac
6
4’-OH Diclofenac
+ESI, 312/231
2C19
S-mephenytoin
40
4’-OH Mephenytoin
-ESI, 233/190
2D6
Bufuralol
7
1’-OH Bufuralol
+ESI, 278/186
2E1
Chlorzoxazone
100
6-OH Chlorzoxazone
-ESI, 184/120
Testosterone
75
6β-OH Testosterone
+ESI, 305/269
Midazolam
1.4
1’-OH Midazolam
+ESI, 342/203
75
50
25
0
-2
-1
0
1
2
-2
75
50
25
-1
0
0
-2
1
-1
0
1
0
2
0
Log C (Tranylcypromine, μM)
Log C (α-Naphthoflavone, μM)
CYP2C19_(+)-N-3-benzylnirvanol
- NAPDH
+ NADPH
100
1
2
75
50
25
160
0
-1
0
1
- NAPDH
+ NADPH
120
80
40
2
-1
-1
0
100
- NAPDH
+ NADPH
80
60
40
20
1
2
-2
-1
0
1
Log C (Quinidine, μM)
Log C (Paroxetine, μM)
CYP2E1_4-Methylpyrazole
CYP3A4_Testosterone_Ketoconazole
CYP3A4_Midazolam_Ketoconazole
% of control activity
75
50
25
0
125
- NAPDH
+ NADPH
100
75
50
25
0
-1
0
1
Log C (4-Methylpyrazole, μM)
2
% of control activity
125
- NAPDH
+ NADPH
100
2
- NAPDH
+ NADPH
100
75
50
25
0
-3
-2
-1
0
0
1
Log C (Sulfaphenazole, μM)
Log C ((+)-N-3-benzylnirvanol, μM)
125
-2
-2
0
-2
25
125
-1
0
1
2
- NAPDH
+ NADPH
100
75
50
25
0
-2
-1
0
1
2
-2
-1
0
1
2
Log C (Metoclopramide, μM)
CYP2E1_Disulfiram
CYP3A4_Testosterone_Troleandomycin
CYP3A4_Midazolam_Troleandomycin
75
50
25
- NAPDH
+ NADPH
100
75
50
25
0
-1
0
1
2
125
- NADPH
100
+ NADPH
75
50
25
125
% of control activity
125
- NAPDH
+ NADPH
100
0
-2
-1
0
1
2
75
50
25
0
-2
Log C (Disulfiram, μM)
- NAPDH
+ NADPH
100
-1
0
1
2
-2
Log C (Troleandomycin, μM)
-1
0
1
2
Log C (Troleandomycin, μM)
3. IC50 Shift of Reversible and Mechanism-Based Inhibitors
Table 2. IC50 shift values of reversible and mechanism-based inhibitors by a 30-min pre-incubation with HLMs
in the presence and absence of NADPH
CYP
IC50 (μM)
Inhibitor
-NADPH
+NADPH
IC50 Shift
(-NADPH/+NADPH)
Inhibitor
Type
0.14
6.0
1.6
0.49
244
5.4
209
0.82
0.79
0.73
0.79
9.1
0.12
0.39
20
1.0
6.1
5.9
0.051
>30
0.13
8.2
0.84
0.77
2.3
0. 10
190
0.42
5.5
0.51
0.12
0.94
0.16
1.7
0.83
0.40
3.2
0.99
1.2
1.3
0.14
0.72
0.25
1.9
0.16
7.7
0.71
4.9
1.3
13
38
1.6
6.5
0.78
4.9
5.2
0.14
0.98
6.3
1.1
5.0
4.6
0.36
>40
0.53
4.2
Reversible
MBI
Reversible
MBI
Reversible
MBI
MBI
Reversible
MBI
Reversible
MBI
MBI
Reversible
Reversible
MBI
Reversible
MBI
MBI
Reversible
MBI
Reversible
MBI
α-Naphthoflavone
Furafylline
Tranylcypromine
8-Methoxypsoralen
Orphenadrine
Thio-TEPA
Phencyclidine
Sulfaphenazole
Tienilic Acid
(+)-N-3-benzylnirvanol
Ticlopidine
Omeprazole
Quinidine
Paroxetine
Metoclopramide
4-Methylpyrazole
Diethyldithiocarbamate
Disulfiram
Ketoconazolea
Troleandomycina
Ketoconazoleb
Troleandomycinb
1A2
2A6
2C9
2D6
3A4
: Testosterone as the probe substrate.
: Midazolam as the probe substrate.
a
b
4. IC50 Shift in Comparison with Co-incubation and Pre-incubation (Fig. 3)
2
100
IC50 co = 0.076 μM
IC50 pre = 0.16 μM
IC50 shift = 0.48
75
50
25
0
-3
-2
-1
0
1
Log C (Ketoconazole, μM)
2
CYP3A4_Testosterone_Troleandomycin
Co-incubation
Pre-incubation
125
100
75
50
25
0
-2
IC50 co = >30 μM
IC50 pre =1.5 μM
IC50 shift = >20
-1
0
1
2
Log C (Troleandomycin, μM)
Fig. 3 IC50 shift of ketoconazole and troleandomycin
on CYP3A4 (testosterone) with co-incubation and
pre-incubation.
CYP3A4_Testosterone_Test Compound A
125
- NADPH
+ NADPH
100
75
50
25
0
-2
IC50 -NADPH = 7.9 μM
IC50 +NADPH = 1.2 μM
IC50 shift = 6.6
-1
0
1
Log C (Test Compound A, μM)
2
CYP3A4_Testosterone_Test Compound A
125
% of control activity
Co-incubation
Pre-incubation
Co-incubation
Pre-incubation
100
75
50
25
0
-2
IC50 co = 14 μM
IC50 pre = 1.3 μM
IC50 shift = 11
-1
0
1
1
Log C (Ketoconazole, μM)
2
-2
-1
0
1
Log C (Ketoconazole, μM)
2
2
Log C (Test Compound A, μM)
Fig. 4 IC50 shift of test compound A on CYP3A4
(testosterone), suggesting test compound A is a MBI.
CYP2D6_Paroxetine
0
-2
3
Log C (Orphenadrine, μM)
CYP2D6_Quinidine
% of control activity
125
% of control activity
-2
50
% of control activity
Fig. 1 Estimation
of IC values of
reversible inhibitors by a
100
100
30-min 75pre-incubation with HLMs
in the presence and
75
absence50 of NADPH, following by 50the addition of the probe
substrates
and a 20-min incubation.
25
25
+ NADPH
100
0
0
- NAPDH
+ NADPH
% of control activity
30
CYP2C9_Sulfaphenazole
125
- NAPDH
50+ NADPH
% of control activity
60
125
% of control activity
% of control activity
90
% of control activity
% of control activity
120
CYP2B6_Orphenadrine
- NAPDH
75
3
CYP2E1_Diethyldithiocarbamate
% o f co n t r o l activ it y
CYP2A6_Tranylcypromine
2
Log C (Omeprazole, μM)
125
% of control activity
1. IC50 Values of Reversible Inhibitors
125
- NAPDH
+ NADPH
100
0
CYP3A4_Testosterone_Ketoconazole
- NAPDH
+ NADPH
1
Log C (Ticlopidine, μM)
125
CYP1A2_α-Naphthoflavone
0
5. Application to Test Compound A using IC50 Shift Approach (Fig. 4)
RESULTS
150
-1
Log C (Tienilic Acid, μM)
2E1
[1] Obach, et al., Drug Metab Dispos 2007, 35:246-255.
[2] Nomeir, et al., Optimization in Drug Discovery: In Vitro Method. p245, 2004.
% of control activity
25
125
- NAPDH
+ NADPH
100
% of control activity
125
- NAPDH
+ NADPH
2C19
3A4
+ NADPH
-2
CYP2D6_Metoclopramide
2B6
1A2
2
CYP2C19_Omeprazole
50
Table 1 Experimental Conditions
LC-MS/MS
(m/z)
1
CYP2C19_Ticlopidine
75
For co-incubation, substrates and inhibitors were warmed at 37˚C for 3 min with HLMs (0.25
mg/ml), and then incubated with NADPH (1 mM) for 20 min. For pre-incubation, only inhibitors were
pre-incubated in the presence of NADPH for 30 min with HLMs, and continually incubated for 20
min after the addition of substrates. CYP reactions were terminated by adding 60% acetonitrile
containing 0.1% formic acid. After centrifugation, the supernatant was injected for the quantitation of
metabolites by LC-MS/MS.
Metabolite
0
CYP2C9_Tienilic Acid
Co-Incubation and Pre-Incubation [2]
Concentration
(μM)
-1
Log C (Phencyclidine, μM)
Log C (Diethyldithiocarbamate, μM)
Substrate
25
0
-2
Log C (Thio-TEPA, μM)
-2
CYP
25
2
Log C (8-MOP, μM)
0
CYP inhibitors were pre-incubated at 37˚C for 30 min with HLMs (0.25 mg/ml) in the presence and
absence of NADPH (1 mM). CYP reactions were initiated by adding probe substrates (final
concentrations were at ~Km) without and with NADPH, incubated at 37˚C for 20 min, and terminated
with 60% acetonitrile containing 0.1% formic acid. After centrifugation, the supernatant was injected
for the quantitation of metabolites by LC-MS/MS.
0
Log C (Furafylline, μM)
100
30-Min Pre-incubation with and without NADPH
CYP2B6_Phencyclidine
0
-2
% of control activity
% of control activity
40
2
0
% of control activity
60
0
-2
125
MATERIALS AND METHODS
80
% of control activity
50
CYP2B6_Thio-TEPA
125
125
Fig.
2 Estimation
of IC50 values
of mechanism-based
inhibitors
- NAPDH
- NAPDH
+ NADPH
100
100
+ NADPH
by
a 30-min
pre-incubation
with HLMs
in the presence and
75
absence of
NADPH, following by 75the addition of the probe
substrates50and a 20-min incubation. 50
- NAPDH
% of control activity
75
0
INTRODUCTION
Mechanism-based inhibition (MBI) on cytochrome P450 (CYPs) reactions leads to irreversible or
quasi-irreversible inactivation of CYPs, and has more clinical impact than reversible inhibition.
Determining that a new chemical entity is a reversible or mechanism-based inhibitor of CYPmediated reactions is critical in drug discovery and development. In this study, known reversible and
mechanism-based inhibitors of eight human CYPs (CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and
3A4) were investigated with pooled human liver microsomes (HLMs) using an IC50 shift method [1].
In the IC50 shift approach, IC50 values are determined using a 30-min pre-incubation with chemical
inhibitors in the presence and absence of NADPH prior to adding substrates, and then the inhibition
type (reversible or mechanism-based) is estimated based on the relative IC50 values under the two
incubation conditions.
CYP2A6_8-MOP
100
- NAPDH
+ NADPH
100
% of control activity
CONCLUSIONS: The IC50 shift approach can be used to differentiate reversible and mechanismbased inhibitors.
CYP1A2_Furafylline
125
% of control activity
METHODS: Determination of IC50 values of known reversible and mechanism-based inhibitors
using human liver microsomes with a 30-min pre-incubation in the presence and absence of
NADPH.
2. IC50 Values of Mechanism-Based Inhibitors
% of control activity
PURPOSE: To investigate the inhibitory difference between reversible and mechanism-based
inhibitors on CYP reactions.
% of control activity
ABSTRACT
CONCLUSION
Mechanism-based inhibitors demonstrated a significant leftward shift in IC50 after pre-incubation in
the presence of NADPH, in comparison with the IC50 after pre-incubation in the absence of
NADPH. In contrast, reversible inhibitors exhibited a rightward shift or no change of IC50 after preincubation with NADPH.
The IC50 shift after pre-incubation in the presence and absence of NADPH can be used to
differentiate reversible (rightward IC50 shift or no change) and mechanism-based (leftward IC50 shift
≥ 4-fold) inhibitors.
The unexpected findings (compared with reports in the scientific literature) that paroxetine
appeared to be a reversible inhibitor of CYP2D6 and omeprazole appeared to be a mechanismbased inhibitor of CYP2C19 require further investigation.
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