Metabolic Stability Assay Using Human Hepatocyte Co-cultures and Integrated Qualitative/Quantitative High Resolution Mass Spectrometry

Alex Zang1, Ragu Ramanathan1, Cornelia Smith2, Caroline Lee2, Helen Shen1, and Zamas Lam1

1: QPS DMPK, Newark, DE; 2: QPS DMPK Hepatic Biosciences, Research Triangle Park, NC

OVERVIEW

Purpose To investigate Sequential Window Acquisition of all Theoretical fragment ion spectra (SWATH™) based integrated qualitative and quantitative (qual/quant) assay for simultaneous metabolic stability and metabolite profiling assessments. Method •An AB Sciex API 5600 TripleTOF mass spectrometer equipped with a Shimadzu Nexera UHPLC was used to analyze human hepatocyte co-cultures using TOF-MS and SWATH scans. • Lorazepam, propranolol, ranitidine, and zoniporide were incubated in 96-well human hepatocyte co-culture plates. Samples were collected at 0, 1, 4, 24, 48, 72, 120, and 168 hours, and buspirone was added as IS. Metabolic stability data were generated using peak-area-ratios of the analyte-to-IS using MultiQuant™ software. The same raw data files were mined for preliminary metabolite information using MetabolitePilot™ software. Results Metabolic Stability: Long term hepatocyte co-cultures enabled measurement of in vitro intrinsic clearance values for low clearance drugs.

Metabolite Identification: Major metabolites were detected following a 7 day incubation with 1 µM drug .

INTRODUCTION

CONCLUSIONS

Using lorazepam, propranolol, ranitidine, and zoniporide as model compounds to acquire metabolic stability and metabolite profile information simultaneously at clinically relevant concentration. This approach is feasible when using high resolution TOF-MS in combination with data independent MS/MS scan. The estimated hepatic clearance values of current study are in line with the observed in vivo CLh values.

Traditional metabolic stability methodology using suspended hepatocyte for drug candidates screening is limited in their ability to accurately predict clinical outcomes. Hepatocyte co-culture platform is a bioengineered, in vitro system with a defined cyto-architecture that provides sustained hepatic functions for at least four weeks1. In this presentation, we investigated human hepatocyte co-cultures model using an integrated qualitative/quantitative high resolution mass spectrometry approach to assess metabolic stability using TOF-MS and Sequential Window Acquisition of All THeoretical fragment ion spectra (SWATH) for non-targeted metabolite MS/MS analysis. The assay was evaluated using propranolol, lorazepam, ranitidine, and zoniporide, at clinically relevant concentration (1 µM) in 96-well format.

METHODS

Metabolic Stability Study Design Substrates: 1. Propranolol (CYP2D6, 1A2, glucuronosyl transferase) 2. Lorazepam (glucuronosyl transferase) 3. Zoniporide (aldehyde oxidase) 4. Ranitidine (FMO, P450, only 6% of dose metabolized; active tubular secretion). Renal

elimination accounts for in vivo clearance. Substrate Concentration: 1 µM • 96-Well HepatoPac™ Micro-patterned co-cultures of Hepatocytes (~5000 cells/well) • 96-Well stromal cells control (~15,000 cells/well) Medium: 64 µL/well Incubation Time: 0, 1, 4, 24, 48, 72, 120, 168 hours Sample Preparation: 60 µL sample aliquots are collected and added to 60 µL of ACN containing IS. The mixtures are vortex-mixed for 2 min, and centrifuged at 3000 rpm for 10 min before analysis. Analysis: Nexera UHPLC-API 5600 (HRMS)

Figure 1. Structures of the Tested Substrates

N

HN

O

Cl

OH

Cl OH

ONH

NO

SNH N

H

NO2

N

NN

N

O

H2N

NH2

Lorazepam (C15H10N2O2Cl2) Exact Mass of [M+H]+ = 321.0188

Propranolol (C16H21NO2) Exact Mass of [M+H]+ = 260.1664

Ranitidine (C13H22N4O3S) Exact Mass of [M+H]+ = 315.1485

Zoniporide(C17H16N6O) Exact Mass of [M+H]+ = 321.1466

Figure 2. Human HepatoPac™ Cell Cultures (Propranolol Incubations)

Pre-Dose Propranolol 168 h treatment

Figure 3. API 5600 Triple TOF Mass Spectrometer

Q0 High Pressure Cell

LINAC® collision cell

40 GHz Multichannel TDC

Detector

Two-stage reflectron

30kHz Accelerator

15 kV Acceleration voltage

Ion compression optics

QJet® Ion Guide

High Frequency Q1

DuoSpray Source

UHPLC System Shimadzu Nexera

UHPLC Column Acquity UPLC BEH C18, 2.1 x 50 mm, particle size 1.7 µm

Column Temperature (oC) 40

Injection Volume (mL) 10

Flow Rate (mL/min) 600

UHPLC Gradient Time (min)

A: 10 mM NH4OAc, pH=5 with Formic

Acid

B: ACN/Formic Acid (100/0.1 V/V)

0.0 95 5

1.0 95 5

3.0 5 95

4.0 5 95

4.1 95 5

5.0 95 5

TOF-MS 100-2000 for 100 ms

SWATH MS/MSALL Range 200-800 Da

SWATH Scans 25 ms per 25 Da window

DP 80 V

CE 35+/-15 V

Total Cycle Time 750 ms

Table 1. Ultra High Pressure Liquid Chromatography and HRMS Parameters

REFERENCE

1. WW Wang, SR Khetani, S Krzyzewski, DB Duignan, RS Obach Assessment of a micro-patterned hepatocyte co-culture system to generate major human excretory and circulating drug metabolites. Drug Metab. Dispos. 2010, 38(10),1900-1905.

2. RJ Riley, DF McGinnity, AP Austin A unified model for predicting human hepatic, metabolic clearance from in vitro intrinsic clearance data in hepatocytes and microsomes Drug Metab. Dispos. 2005, 33(9), 1304-1311.

3. D Dalvie, CH Zhang, WH Chen, T Smolarek, RS Obach, CM Loi Cross-species comparison of the metabolism and excretion of Zoniporide: contribution of aldehyde oxidase to interspecies differences. Drug Metab. Dispos. 2010, 38(4), 641-654.

RESULTS

Figure 4. Integrated Qualitative and Quantitative Metabolic Stability Analysis

Qua

litat

ive

Bioa

naly

sis

Qua

ntita

tive

Bioa

naly

sis

Full Scan HRMS Data + SWATH

Advantages Using UHPLC-Q-TOF for Quantitative and Qualitative Bioanalysis

No need for optimization or decide on a fragment, using the generic acquisition conditions

Full scan MS preserves all the information about the sample (drug, metabolites, dosing vehicle, degradants, biomarkers, etc.)

HRMS provides opportunities to use mass defect filter (MDF), isotope pattern filtering, background subtraction, etc. for metabolite detection.

Provides an option for acquiring quantitative and qualitative information simultaneously.

Figure 5. Metabolic Stability Results

0.001

0.01

0.1

1

0 2000 4000 6000 8000 10000 120000.00001

0.0001

0.001

0.01

0.1

1

10

0 2000 4000 6000 8000 10000 12000

0.1

1

10

0 2000 4000 6000 8000 10000 120000.0001

0.001

0.01

0.1

1

10

0 2000 4000 6000 8000 10000 12000

Pak

Area

Rat

io (P

AR)

Time, min

Figure 6. Propranolol (1 µM) Metabolite Formation (MetabolitePilot™ Software)

Figure 7. Metabolite Formation Time Courses of Four Drugs from Single Incubations

-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180Time, Hour

0.0e0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

Are

a

72.00 168.0024.00

Oxidation and Sulfate Conjugation, m/z=356.1153, RT=2.32 minOxidation, m/z=276.1617, RT=2.51 minGlucuronide Conjugation, m/z=436.1959, RT=2.60 minParent, m/z=260.1668, RT=2.74 min

-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180Time, Hour

0.0e0

5.0e3

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

Are

a

Glucuronide Conjugation, m/z=497.0507, RT=2.59 minParent, m/z=321.0196, RT=2.91 min

-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180Time, Hour

0.0e0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

5.0e5

5.5e5

6.0e5

6.5e5

7.0e5

7.5e5

8.0e5

8.5e5

9.0e5

9.5e5

Are

a

Loss of CH2, m/z=301.1325, RT=1.12 minOxidation, m/z=331.1432, RT=1.28 minParent, m/z=315.1491, RT=1.31 min

-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180Time, Hour

0.0e0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

5.0e5

5.5e5

6.0e5

6.5e5

7.0e5

7.5e5

8.0e5

8.5e5

9.0e5

Are

a

72.00

24.00 48.00120.00

168.00

4.00

Oxidation, m/z=337.1426, RT=2.27 minOxidation, m/z=337.1417, RT=2.35 minParent, m/z=321.1470, RT=2.48 min

Propranolol

Ranitidine

Zoniporide Mets: two oxidation metabolites

Lorazepam

Propranolol Mets: oxidation+sulfate, oxidation, glucuronide

Ranitidine Mets: demethylation,

oxidation

Lorazepam Met: glucuronide

Zoniporide

Estimated Hepatic Clearance Values CLh

(mL/min*kg) of Current Study

In Vivo Hepatic Clearance Values CLh

(mL/min*kg) From Literature

Lorazepam 5.5 1.12

Propranolol 16.8 16.112

Ranitidine 2.1 2.92

Zoniporide 12.4 213