Metabolic Stability Assay Using Human Hepatocyte Co-cultures … · 2014. 12. 5. ·...
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Metabolic Stability Assay Using Human Hepatocyte Co-cultures and Integrated Qualitative/Quantitative High Resolution Mass Spectrometry Alex Zang 1 , Ragu Ramanathan 1 , Cornelia Smith 2 , Caroline Lee 2 , Helen Shen 1 , and Zamas Lam 1 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 CL h 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 weeks 1 . 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 H N O Cl OH Cl OH O N H N O S N H N H NO 2 N N N N O H 2 N NH 2 Lorazepam (C 15 H 10 N 2 O 2 Cl 2 ) Exact Mass of [M+H] + = 321.0188 Propranolol (C 16 H 21 NO 2 ) Exact Mass of [M+H] + = 260.1664 Ranitidine (C 13 H 22 N 4 O 3 S) Exact Mass of [M+H] + = 315.1485 Zoniporide(C 17 H 16 N 6 O) 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 ( o C) 40 Injection Volume (mL) 10 Flow Rate (mL/min) 600 UHPLC Gradient Time (min) A: 10 mM NH 4 OAc, 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/MS ALL 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 Qualitative Bioanalysis Quantitative Bioanalysis 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 12000 0.00001 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 12000 0.0001 0.001 0.01 0.1 1 10 0 2000 4000 6000 8000 10000 12000 Pak Area Ratio (PAR) 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 90 100 110 120 130 140 150 160 170 180 Time, 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 Area 72.00 168.00 24.00 Oxidation and Sulfate Conjugation, m/z=356.1153, RT=2.32 min Oxidation, m/z=276.1617, RT=2.51 min Glucuronide Conjugation, m/z=436.1959, RT=2.60 min Parent, m/z=260.1668, RT=2.74 min -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 Time, 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 Area Glucuronide Conjugation, m/z=497.0507, RT=2.59 min Parent, m/z=321.0196, RT=2.91 min -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 Time, 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 Area Loss of CH2, m/z=301.1325, RT=1.12 min Oxidation, m/z=331.1432, RT=1.28 min Parent, m/z=315.1491, RT=1.31 min -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 Time, 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 Area 72.00 24.00 48.00 120.00 168.00 4.00 Oxidation, m/z=337.1426, RT=2.27 min Oxidation, m/z=337.1417, RT=2.35 min Parent, 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 CL h (mL/min*kg) of Current Study In Vivo Hepatic Clearance Values CL h (mL/min*kg) From Literature Lorazepam 5.5 1.1 2 Propranolol 16.8 16.11 2 Ranitidine 2.1 2.9 2 Zoniporide 12.4 21 3
Transcript of Metabolic Stability Assay Using Human Hepatocyte Co-cultures … · 2014. 12. 5. ·...
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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 NH
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
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0 2000 4000 6000 8000 10000 120000.00001
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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
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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
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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
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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
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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
Metabolic Stability Assay Using Human Hepatocyte Co-cultures and Integrated Qualitative/Quantitative High Resolution Mass Spectrometry
