Transcript ON DECK INCUBATION AND 384-WELL FORMAT SIMPLIFY AND

ON DECK INCUBATION AND 384-WELL FORMAT SIMPLIFY AND INCREASE THROUGHPUT FOR HUMAN LIVER MICROSOMAL SCREENING
IN AN HT-ADME SCREENING LABORATORY
M. Snyder, C. Taylor, J. Janiszewski & K. Whalen - PDM, Pfizer Global Research and Development, Groton Laboratories, Groton, CT 06340
Automation/Bioanalytical:
 Caliper Life Sciences Sciclone ALH3000
and Beckman Coulter Biomek FX
workstations equipped with 384-channel
disposable tip array were used to
perform all liquid handling steps.
 An AB/Sciex API3000 triple quadrupole
mass spectrometer, operating in
selected reaction monitoring (SRM)
mode, was used for analyte and internal
standard detection.
• Results
 Validation results were assessed
relative to published clearance values.
 Key learning’s:
Rapid Dispense (100 µl /sec)
On-deck Incubation
Scheduling with Excel Macro
P450 concentration: 0.25 µM (0.76 mg protein/mL)
Compound substrate concentration: 1 µM
Co-Factor utilizes NADPH regeneration system
w/ 1 mM MgCl2
Buffer concentration: 100 mM KPO4, pH 7.4
Time-points: 0, 5, 10, 20, 30 and 60 minutes
Internal standard used to ensure LC/MS/MS
performance and reproducibility
Liquid handling performed on Caliper Life Sciences
Sciclone ALH3000 or Beckman Coulter Biomek FX
workstation
2.
Microsomes
& Cofactor
4.
y = 1.2534x - 2.1081
2
R = 0.8603
250.00
200.00
150.00
y = 1.2401x - 2.5148
2
R = 0.8171
100.00
Compound
y = 0.9416x + 1.3742
2
50.00
ALH FX
0.00
0.00
50.00
100.00
150.00
200.00
250.00
D
Figure 4. Intrinsic clearance values for 12
compounds (n=96) run on both automated
workstations were similar to reported values.
B
A
B
Figure 3. FX Deck Layout. (A) 3 X 4 Peltier heating ALP, (B) Recirculating
reservoir containing cold acetonitrile and IS, (C) Microsome reservoir,
(D) 384-well tip-wash station, (E) Extra deck space where crashed plates
are stacked off of heat not shown.
34 µl/sec
A+B
100 µl/sec
Figure 6. Non-contact
mixing.
50.00
100.00
150.00
Reaction
Temp. (°C)
Mecour
Peltier
Average
35.01
31.4
Range
34.5 – 35.7
31.4 – 36.8
STDEV
0.295
1.13
Table 1. Measured
temperature variation for
on-deck incubation
hardware.
Microsome Assay_12_11_2006_11_16_33.xls
12_11_2006
Start Time End Time
60 Minutes no drug, Cmpnd A
10:09:21
60 Minutes no drug, Cmpnd B
10:09:40
60 Minutes no CoF, Cmpnd A
10:10:37
60 Minutes no CoF, Cmpnd B
10:10:56
60 Minutes CoF, Cmpnd A
10:11:28
60 Minutes CoF, Cmpnd B
10:11:46
30 Minutes CoF, Cmpnd A
10:12:16 10:42:12
30 Minutes CoF, Cmpnd B
10:12:34 10:42:31
20 Minutes CoF, Cmpnd A
10:13:04 10:33:00
20 Minutes CoF, Cmpnd B
10:13:24 10:33:19
10 Minutes CoF, Cmpnd A
10:13:53 10:23:48
10 Minutes CoF, Cmpnd B
10:14:12 10:24:07
5 Minutes CoF, Cmpnd A
10:14:25 10:19:20
5 Minutes CoF, Cmpnd B
10:14:42 10:19:38
0 Minutes CoF, Cmpnd A
10:17:13
0 Minutes CoF, Cmpnd B
10:18:01
Actual Incubation
Requested Incubation timer flags cell counters
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0:59:45
1
17
17
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0:59:45
1
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0:59:45
1
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0:59:45
1
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0:59:45
1
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0:59:45
1
0:29:56
0:29:45
0:29:57
0:29:45
0:19:56
0:19:45
0:19:55
0:19:45
0:09:55
0:09:45
0:09:55
0:09:45
0:04:55
0:04:45
0:04:56
0:04:45
10:17:13
0:00:00
10:18:01
0:00:00
Table 2. Excel scheduling macro used with
Sciclone for time-point incubations. Written by
Jim Batchelor, Caliper Life Sciences.
Conclusions: Key Learning’s
References
3.
Incubate @
37°C
0, 5, 10, 20, 30, 60 min.
Figure 1. 27.8 µl assay.
1. Riley Robert J; McGinnity D F; Austin R P. A unified model for predicting
human hepatic, metabolic clearance from in vitro intrinsic clearance data in
hepatocytes and microsomes. Drug Metab and Dispos (2005), 33(9), 130411.
2. Obach, R. Scott. Prediction of human clearance of twenty-nine drugs from
hepatic microsomal intrinsic clearance data: an examination of in vitro halflife approach and nonspecific binding to microsomes. Drug Metab and
Dispos (1999), 27(11), 1350-1359.
200.00
Figure 5. Different pipetting techniques and ondeck incubation provided similar Cl int values on
either workstation.
C
ACN (Protein
Precip.)
0.00
0.00
Sciclone ALH 3000 Clint
Experimental Clint
75 µl
Assay (8)
100.00
50.00
25 µl
2.8 µl
150.00
R = 0.9226
A
1.
200.00
Nicardipine
300.00
Midazolam
Propafenone
250.00
Prednisone
Figure 2. Sciclone Deck Layout. (A) Two 6-position Mecour heating
blocks, (B) Recirculating reservoir containing cold acetonitrile and IS,
(C) Microsome reservoir, (D) 384-well tip-wash station, (E) Extra deck
space where crashed plates are moved off of heat.
350.00
Verapamil
A
Diclofenac
D
C
Desipramine
Propranolol
Dilitiazem
Methods: Microsome Assay Specifics
B
 The experimental results correlated well to the literature values (Figure 4). The larger discrepancies may
be attributed to the differing incubation conditions (e.g. protein concentration).
 Little difference in workstations (Figure 5).
 The combination of 384-well format and automated liquid handling allows for 720 test compounds to be
assayed in a single morning. This format equates to a potential 3-day throughput of over 4300
compounds.
 Further throughput gains are restricted by LC/MS/MS analysis. (i.e. 4300 compounds = 43,000 LC
injections)
 The large capacity allows us to maintain discovery chemistry’s capacity of over 2000 compounds per
week.
Quinidine
Amitriptyline
In drug discovery, the number of chemical compounds
requiring in-vitro ADME screening is ever increasing.
To keep pace with compound submissions, new
methods must be developed to increase the throughput
of current in-vitro ADME assays. Herein, we present our
group’s efforts towards increasing throughput of the
Human Liver Microsome metabolic stability assay using
a compressed 384-well format and an automated liquid
handler.
Diazepam
• Methods
Biology:
 Twelve compounds with a broad range
of clearance rates and P450 pathways
were chosen to validate assay1,2.
 Eight plates were generated per 384well master plate: a matrix blank; 0, 5,
10, 20, 30, 60 minute timepoints; and a
60 minute no co-factor control.
 Assay volume: 27.8 µl.
E
Results
Biomek FX Clint
• Introduction
 To develop an automated 384-well
microsomal metabolic stability assay to
support rapid screening of 2,000
discovery compounds per week.
Introduction
Literature Clint
Overview
Rapid Dispense/ Non-contact Mixing
100 µl/sec - Savings in Tips, Time, & Deck Space (Figure 6).
On-Deck Incubation @ 37°C
Savings in Hardware/ Human Intervention (Table 1).
Easy Scheduling with Excel Macro (Table 2).
27.8 µl Assay in 384 well plate
Savings in Microsomes - 720 Compounds in 3 hours!
250.00
• Rapid Dispense Speed
– 100 ul/sec
34 ul/sec
100 ul/sec
• Deck space, tips ($$$$)
• On-deck Incubation
– Mecour Thermal Blocks
Success:
1. Automated dilution during Mic prep
2. 1 hour unattended completion of assay