A New Approach to Time-Dependent Cytochrome P450 Inhibition Assay
®
Using the Echo Liquid Handler
Sammy
1
Datwani ,
Joseph
1
Barco ,
Sean Xiang
2
Wu ,
Wentao
2
Zhang ,
and Ji Young
Sunnyvale, CA. www.labcyte.com 2Quintara Discovery, South San Francisco, CA. www.quintaradiscovery.com 3Celsis IVT, Baltimore, MD. www.celsisivt.com
Abstract
Early assessment of the ADMET (Absorption, Distribution,
Metabolism, Excretion and Toxicity) properties of drug
candidates has become a critical part of modern drug discovery.
The frequent failure of compounds due to poor ADMET
properties
necessitates
earlier
profiling
of
ADMET
characterizations. As a large number of compounds are
screened for ADMET properties against various cytochrome
P450 (CYP450) enzymes on a regular basis, miniaturization and
automation of ADMET assays have become a top priority to
manage the cost and labor. The time-dependent CYP450
inhibition assay is one of the most important ADMET assays
which identifies drug candidates that may have sub-optimal
pharmacokinetic properties or potential for undesirable drugdrug interactions. Here, we present a miniaturized, simplified
and automated method using the Echo liquid handler to evaluate
time-dependent CYP inhibition potential with RapidFire/MS
(RF/MS). The results from the new method generate fast,
reliable and accurate IC50 results while reducing reagent costs
by 90%.
3
Lee
Miniaturized time-dependent CYP450 inhibition assay
Results
The purpose of this experiment was to evaluate a time-dependent CYP450 inhibition
assay in a miniaturized format. Two methods were utilized:
1. All reagents transferred with the Echo 555 liquid handler.
2. Compounds transferred with the Echo 555 liquid handler; all other reagents
transferred with the Echo 525 liquid handler.
IC50 values and timing generated from both instruments were compared.
Miniaturized and simplified time-dependent CYP450 inhibition assays were developed
with Labcyte Echo liquid handlers. CYP3A4 inhibition profiles from four known
inhibitors were tested. Ketoconazole, a known and potent CYP3A4 inhibitor, showed
no change in IC50 (ΔIC50 = 1) during incubation. Mibefradil, Verapamil, and
Mifepristone, three known time-dependent inhibitors of CYP3A4, all showed greater
than five fold changes during incubation in the miniaturized time-dependent inhibition
assay with the Echo liquid handlers. CYP1A2 inhibition profiles from two reference
inhibitors were also evaluated. Both compounds showed greater than 3 fold changes
in IC50 during in vitro incubation. Data generated in miniaturized and simplified timedependent CYP inhibition assay showed consistent results when compared with
published literature values.
Methods
Ketoconazole, Mibefradil, Mifepristone, Verapamil, Furafylline and Fluvoxamine were weighed out and
dissolved in DMSO to make 50 mM stock solutions. Sixteen compound concentrations were generated
in DMSO by direct dilution using the Echo® Dose-Response software application. 10 nL diluted
compounds were transferred to two 384-well Greiner microplates with the Echo 555 liquid handler.
50 mM potassium phosphate buffer, pH 7.4 was made by combining potassium phosphate monobasic
solution and potassium phosphate dibasic solution. 1 mM NADPH was dissolved in the 50 mM
potassium phosphate buffer. Both potassium phosphate buffer and NADPH buffer were kept in a 37°C
water bath. HLM were diluted to 1.5 mg/mL in both potassium phosphate buffer and 1 mM NADPH
buffer. 60 µL HLM in potassium phosphate buffer were dispensed into one column and 60 µL HLM in
NADPH buffer were dispensed into another column of a 384-well Echo qualified polypropylene plate.
Inhibition profiling of the six reference compounds and transfer speed were also
compared between Echo 555 and 525 liquid handler. Table 3 demonstrates that IC50
values generated from both Echo liquid handlers showed almost identical inhibition
profiling. When assay-ready plates were readily available, the Echo 525 liquid
handler can be used to transfer various aqueous fluid with increased speed.
Echo 555
Echo 525
Echo 555 Method: The Echo 555 liquid handler was used with the Echo® Plate Reformat software
application to transfer 2 µL HLM in potassium phosphate buffer with or without 1 mM NADPH. The
entire HLM transfer took approximately 16 minutes per 384-well microplate.
Echo 525 Method: The Echo 525 liquid handler was used with the Echo Plate Reformat software
application to transfer 2 µL HLM in potassium phosphate buffer with or without 1 mM NADPH. The
entire HLM transfer took approximately four minutes per 384-well microplate.
120
- NADPH
120
100
+ NADPH
100
% Inhibition
1Labcyte Inc.,
Brent
1
Eaton ,
% Inhibition
Jing
1
Wang ,
80
60
40
Volume
increment
Features
and
Capabilities
•
•
•
•
Echo 555
Echo 525
2.5 nL
25 nL
Compound dilution
Compound addition
Aqueous transfer
One liquid handler
enabled for every
transfer
• Rapid transfer for
broad range of
aqueous fluids
into assay-ready
plates
0
1
IC50 (nM)
2
3
4
0
5
1
2
3
4
5
Log [Verapamil ] nM
- NADPH
+NADPH
 IC50
25.53
34.26
1
IC 5 0 (nM )
- NADPH
+NADPH
15848
424.6
 IC 50
37
120
- NADPH
100
+ NADPH
80
60
40
20
0
1
2
3
+ NADPH
80
60
40
0
4
0
Log [Mibefradil ] nM
1
2
3
4
5
log [ Mifepristone ] nM
- NADPH
+NADPH
 IC 50
1809
232
8
IC 50 (nM)
- NADPH
+NADPH
7943
145.6
 IC 50
54
100
-NADPH
80
60
40
20
- NADPH
+ NADPH
80
+NADPH
% Inhibition
CYP1A2
- NADPH
100
20
0
100
120
% Inhibition
% Inhibition
CYP3A4
IC 50 (nM)
60
40
20
0
0
1
2
3
4
0
5
0
IC 50 (nM )
HLM
Transfer
- NADPH
+NADPH
83.36
25.95
1
2
3
4
5
log [Furafylline] nM
log [Fluvoxamine] nM
 IC 50
3
IC 5 0 (nM )
- NADPH
+NADPH
10892
148.1
~ 16 minutes
 IC 50
73
~ 4 minutes
Table 3. IC50 generated from Echo 555 and 525 liquid handler showed similar
Inhibition profiling. While both liquid handlers can transfer all reagents, the transfer of
HLM was significantly faster on the Echo 525 system.
Figure 1. Workflow comparison between conventional and miniaturized timedependent CYP450 inhibition assay. The entire assay procedure has been simplified
by using the Echo liquid handlers in comparison with the conventional assay.
In comparison with the conventional assay, miniaturized time-dependent CYP450
inhibition assay offers cost savings when performing compound inhibition profiling
against various CYP450 enzymes.
Compounds, HLM and substrate volume
requirements are drastically reduced in the miniaturized format with the Echo liquid
handler. Table 2 lists the reduction in material used in the conventional assay vs. the
miniaturized assay.
Compounds
HLM
Substrate
Conventional
Echo 5XX
1 µL
100 µL
90 µL
10 nL
2 µL
20 µL
X-Fold
Reduction
100
50
4.5
Table 2. Reagents are significantly reduced in the miniaturized time-dependent
CYP450 inhibition assay.
Table 1. Echo 555 and 525 liquid handlers each represent unique features for
different assay setup.
40
Log [ketoconazole] nM
% Inhibition
Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) properties of
drug candidates have been widely adopted as an integral part of modern drug
discovery for compound advancement and clinical candidate selection. They are
critical factors in predicting pharmacokinetic behavior. One of the most important
ADMET assays is time-dependent CYP450 inhibition assay. Compounds with timedependent inhibition are thought to cause more serious drug-drug interactions. It is
inevitable that a large number of compounds will be screened in the time-dependent
inhibition assays against many CYP450 enzymes. The ability to miniaturize timedependent CYP450 inhibition assays will be a valuable tool in drug discovery
research.
The Labcyte Echo liquid handler revolutionizes liquid transfer by using acoustic
energy to eject fluids. The Echo liquid handlers transfer low nanoliter droplets
repeatedly, so precision and accuracy are consistent over a larger volume range.
Large volume transfer is achieved by transferring several hundred droplets per
second. Transfer is non-contact and tipless, with increased cost savings from
elimination of tip costs and washing fluids. The Echo liquid handler also allows for
assay miniaturization to previously unattainable volumes.
The Echo 525 liquid handler is the newest model of the Echo liquid handler, designed
for rapid transfer of many biochemical and genomics reagents (buffers, proteins,
glycerol, etc.) for assay assembly.
The Echo 555 liquid handler, transfers in 2.5 nL volume increments and is optimized
for compound dilution and reagent addition. The Echo 555 liquid handler alone can be
used for the entire assay assembly. The Echo 525 liquid handler transfers in 25 nL
volume increments and can be used for rapid transfer of a broad range of aqueous
reagents into assay ready plates. Table 1 summarizes the unique characteristics of
the Echo 555 and 525 liquid handlers
All Echo liquid handler models can be used to transfer from any source well to any
destination well, without the need for user calibration. Dynamic Fluid Analysis™ is a
property utilized in the Echo liquid handlers to enable real time optimization of reagent
transfer, even for reagents never before transferred with the platform.
60
0
-1
Introduction
80
20
20
0
The HLM and compound mixtures were then incubated in a 37°C water bath for 30 minutes. During the
incubation, 5 µM 9-Amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate and 30 µM testosterone, all
FDA-acceptable substrates, were made up in 1 mM NADPH buffer and kept in a 37°C water bath. At
the end of the 30 minute incubation, 20 µL of 5 µM 9-Amino-1,2,3,4-tetrahydroacridine hydrochloride
hydrate were dispensed into one of the 384-well destination microplates. 20 µL of 30 µM testosterone
were dispensed into another 384-well destination microplate. This reaction was further incubated for 15
minutes. At the end of the 15-minute incubation, 20 µL acetonitrile was added to quench the reaction.
Bucetin was present in the quench solution as an internal standard. Quenched samples were cooled to
4°C, and centrifuged at 2500xg for 15 minutes.
The assay plates were then used directly for sample injection by the Rapid-Fire/MS system. Formation
of 1-hydroxy-tacrine and 6β-hydroxytestosterone were monitored by mass spectroscopy.
- NADPH
+ NADPH
Summary
•
•
•
•
•
Conventional TDI ADME Assays can be run in a
miniaturized format to reduce compound and reagent
volumes.
Both the Echo 525 and Echo 555 liquid handlers can
transfer all reagents required to miniaturize the TDI assay
format.
The Echo 555 liquid handler can be used to transfer low
nanoliter volumes of compounds and be utilized in an
assay-ready plate formatting workflow.
The Echo 525 liquid handler can rapidly transfer HLM to
generate TDI data.
Both Echo platforms can be used in tandem in laboratories
that prepare compounds for assay ready plates and fully
assemble biochemical assays.