on deck incubation and 384-well format simplify and increase throughput for human liver microsomal...

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

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.

Results• Introduction

To develop an automated 384-well microsomal metabolic stability assay to support rapid screening of 2,000 discovery compounds per week.

• 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 384-well 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. 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 IncubationScheduling with Excel Macro

P450 concentration: 0.25 µM (0.76 mg protein/mL)Compound substrate concentration: 1 µMCo-Factor utilizes NADPH regeneration system

w/ 1 mM MgCl2Buffer concentration: 100 mM KPO4, pH 7.4Time-points: 0, 5, 10, 20, 30 and 60 minutesInternal standard used to ensure LC/MS/MS

performance and reproducibilityLiquid handling performed on Caliper Life Sciences

Sciclone ALH3000 or Beckman Coulter Biomek FX workstation

Methods: Microsome Assay Specifics

Overview Introduction

0 MIN

5 MIN

10 MIN

20 MIN

30 MIN

60 MIN

60 MIN

NO CoF

TIP BOX

ACN & IS

A

BC

D

E

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.

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.

References1. 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), 1304-11.

2. Obach, R. Scott. Prediction of human clearance of twenty-nine drugs from hepatic microsomal intrinsic clearance data: an examination of in vitro half-life approach and nonspecific binding to microsomes. Drug Metab and Dispos (1999), 27(11), 1350-1359.

Compound Assay (8)2.8 µl

Microsomes& Cofactor

25 µl

ACN (ProteinPrecip.)

75 µl1.

Incubate @ 37°C

0, 5, 10, 20, 30, 60 min.

2.

Conclusions: Key Learning’sRapid 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!

34 µl/sec 100 µl/sec

3.

4.

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.

D

C

A

B

Figure 1. 27.8 µl assay.

Figure 4. Intrinsic clearance values for 12 compounds (n=96) run on both automated workstations were similar to reported values.

y = 0.9416x + 1.3742

R2 = 0.9226

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Sciclone ALH 3000 Clint

Bio

me

k F

X C

l int

y = 1.2534x - 2.1081

R2 = 0.8603

y = 1.2401x - 2.5148

R2 = 0.8171

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Experimental Clint

Lit

era

ture

Cl in

t

ALH FX

Figure 5. Different pipetting techniques and on-deck incubation provided similar Cl int values on either workstation.

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. Table 2. Excel scheduling macro used with

Sciclone for time-point incubations. Written by Jim Batchelor, Caliper Life Sciences.

Figure 6. Non-contact mixing.

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Microsome Assay_12_11_2006_11_16_33.xls12_11_2006 Start Time End Time Actual Incubation Requested Incubation timer flags cell counters

60 Minutes no drug, Cmpnd A 10:09:21 ################# 0:59:45 1 17 1760 Minutes no drug, Cmpnd B 10:09:40 ################# 0:59:45 160 Minutes no CoF, Cmpnd A 10:10:37 ################# 0:59:45 160 Minutes no CoF, Cmpnd B 10:10:56 ################# 0:59:45 1

60 Minutes CoF, Cmpnd A 10:11:28 ################# 0:59:45 160 Minutes CoF, Cmpnd B 10:11:46 ################# 0:59:45 130 Minutes CoF, Cmpnd A 10:12:16 10:42:12 0:29:56 0:29:4530 Minutes CoF, Cmpnd B 10:12:34 10:42:31 0:29:57 0:29:4520 Minutes CoF, Cmpnd A 10:13:04 10:33:00 0:19:56 0:19:4520 Minutes CoF, Cmpnd B 10:13:24 10:33:19 0:19:55 0:19:4510 Minutes CoF, Cmpnd A 10:13:53 10:23:48 0:09:55 0:09:4510 Minutes CoF, Cmpnd B 10:14:12 10:24:07 0:09:55 0:09:455 Minutes CoF, Cmpnd A 10:14:25 10:19:20 0:04:55 0:04:455 Minutes CoF, Cmpnd B 10:14:42 10:19:38 0:04:56 0:04:450 Minutes CoF, Cmpnd A 10:17:13 10:17:13 0:00:000 Minutes CoF, Cmpnd B 10:18:01 10:18:01 0:00:00

Success & Key Learning's from Microsome Assay Automation• Rapid Dispense Speed– 100 ul/sec

• Deck space, tips ($$$$)

• On-deck Incubation– Mecour Thermal Blocks

• Temp range 5.4 vs. 1.2°C

• Easy Scheduling– Excel Macro

34 ul/sec 100 ul/sec

Success:1. Automated dilution during Mic prep2. 1 hour unattended completion of assay