High-Throughput LDTD384-MS/MS for Drug Metabolism and Pharmacokinetic Studies 1Sébastien
Gagné; 2Patrice Tremblay; 1Francis Foczeny; 1Robert Houle; 1Eric Langlois; 1Kevin Bateman; 2Pierre Picard 1Merck
Canada, Kirkland, QC, Canada 2Phytronix Technologies, Quebec, QC, Canada
58th ASMS Conference on Mass Spectrometry Salt Palace Convention Center Salt Lake City, Utah
Background •
Drug discovery – Process by which potential drugs are discovered through new chemical entities (NCE) – Involve several fields • One of them Æ Drug Metabolism and Pharmacokinetics (DMPK)
•
DMPK criteria (assessed using mass spectrometry) – – – – – – – – – – –
Pharmacokinetics Drug-Drug Interaction Metabolic stability Metabolic profiles (in vitro / in vivo) Distribution and route of elimination Plasma Protein Binding Permeability Transporter Blood / Plasma PK prediction Phenotyping
Can we differentiate compounds faster?
Introduction • The LDTD source – – – – – –
Developed by Phytronix Technologies (Québec, QC, Canada) Indirect thermal desorption by a laser diode (980 nm, 20 W) Atmospheric pressure chemical ionization (APCI) Atmospheric pressure, fit with any mass spectrometer Minimal sample prep, no LC separation No solvent, no matrix application
Sample Plate Laser Diode
Gas Line Orifice
lens
Peltier Cooler
Laser diode
Piston x-y stage
Discharge Needle
Schematic of the LDTD Source
LazWell Sample Plate Carrier Gas Transfer Tube Mass Spectrometer Inlet
IR Laser Beam Piston Head
Piston
Corona Discharge Needle
LDTD Source on Thermo-Quantum LDTD source
10-plate sample loader
Source power supply & gas controller
Experimental details • Instrumentation – LDTD source + Thermo Quantum – LazWell sample plate • • • • • •
NEW Standard 384-well plate format, disposable Well: proprietary stainless steel alloy 1-2.5μL of sample per well (no matrix) No carryover: each sample has its own well Amenable to robotic sample preparation systems Current well design: hexagonal
• Experimental condition set up
LazWell plate
– The SRM conditions of each analyte/internal standard were optimized directly by LDTD-MS using the Quantum Tune page (positive mode) • 1 μL of each standard was spotted on the LazWell plate, dried, and laser power/duration, carrier gas and SRM conditions were optimized individually.
– Carrier gas for LDTD: compressed air, 35°C, 3 L/min
LDTD384-MS/MS – Over all performances HPLC-MS/MS (API4000)
LDTD384-MS/MS (Quantum)
Run time = 5 minutes (300 seconds) sample-to-sample
Run time = 12 secondes sample-to-sample
100
Lazer power (%)
% organic solvent
25x faster
80 60 40 20 0 0
1
2
3
Time (min)
4
5
50 40 30 20 10 0 0
2
4
6
Time (sec)
8
10
LDTD384-MS/MS – Over all performances 77 mins
RT: 0.12 - 75.90 100
NL: 4.06E7 TIC F: + p APCI SRM ms2
[email protected] [100.160-100.260] MS Clomiphene_1uL_fullpl ate
95 90 85 80
70 65
95
60 55
NL: 4.06E7 TIC F: + p APCI SRM ms2
[email protected] [100.160-100.260] MS Clomiphene_1uL_fullpl ate
CV = 9.3%
90
50 45
85
40 35
80
30 25
75
20 15 10
70
5 0 10
20
30
40 Time (min)
50
60
65
70
Relative Abundance
Relative Abundance
n = 384
RT: 9.99 - 19.97 100
75
Clomiphene (1µM)
60 55 50 45 40 35 30 25 20 15 10 5 0 10
11
12
13
14
15 Time (min)
16
17
18
19
LDTD384-MS/MS – Over all performances 100µM
9000
5000000
•Plasma samples spiked and quenched with 3 volumes of ACN and diluted with 5 volume of MeOH + EDTA
200nM 8000
•LOD ~ 5nM
7000
•5 order of magnitude
6000
3000000
Ciprofloxacin Y = -0.00320281+0.000547676*X R^2 = 0.9962 W: 1/X
5000
60
4000
55
3000
50
2000
20nM
1000
5nM 0 0
100
200
300
400
R2 = 0.992
45
50µM Area Ratio Area ratio
Intensity
Intensity
4000000
500
Concentration (uM)
2000000
40 35 30 25
25µM 20
1000000
15 10
10µM 0.2µM 2.5µM
0 0
200
400
600
800
Concentration (uM)
5 0
1000
1200
0
20000
40000
60000
Concentration (uM)
80000
100000
Applications • Application #1: Drug-drug interaction – Time-Dependent Inhibition • Ability of a drug to decrease CYP activities
• Application #2: Metabolic Stability/Intrinsic Clearance – In vitro tool used to predict the stability of a compound
• Application #3: Pharmacokinetics – Explore what the body does to the drug
Application #1 – Time Dependent Inhibition • Kobs Î Test compound at 10uM & 50uM Î Pre-incubation: 0’, 5’, 10’, 15’, 20’ OH
Screening approach OH
X
CYP3A4 O
O
INHIBITOR
OH
6β-hydroxy-testosterone
Testosterone
Tim e dependent inhibition
LN (% activity) (vs MeOH)
5.0
No TDI
4.0 3.0
TDI if slope k > 0.015min-1
2.0 1.0 0.0 0
5
10
15
20
25
Pre-incubation time (min)
30
Application #1 – Time Dependent Inhibition Î Known to cause TDI
Mifepristone
HPLC-MS/MS (API4000)
LDTD384-MS/MS (Quantum)
Tim edependent dependent Inhibition inhibition of efipristone Time ofmMifepristone
Time ofmMifepristone Tim edependent dependentInhibition inhibition of efipristone MeOH
50uM
4.0
10uM
3.0 2.0
y = -0.0963x + 3.9541 y = -0.0938x + 4.3285 R2 = 0.9734 R2 = 0.9733
1.0 0.0 0
5
10
15
20
25
Pre-incubation time (min)
30
LN (% activity) (compared to MeOH)
LN (% activity) (compared to MeOH)
MeOH 5.0
5.0
50uM
4.0
10uM
3.0 2.0
y = -0.0754x + 3.9783 y = -0.0814x + 4.3958 R2 = 0.9803 R2 = 0.9225
1.0 0.0 0
5
10
15
20
25
Pre-incubation time (min)
Comparable results by HPLC-MS/MS and LDTD384-MS/MS
30
Application #2 – Metabolic stability • Hepatocytes – Cells involved in the detoxification, modification and excretion of exogenous substances liver
Add Compound
Collagenase Hepatocytes in suspension
(1 μM)
Incubate @ 37oC 95/5 O2/CO2
• Liver microsomes – Concentrated Cytochrome P450 being a valuable tool for investigating the metabolism of compounds liver
Incubate @37°C Sub-cellular Supplemented Fraction preparation Liver Microsomes w / cofactors
% Parent remaining
Application #2 – Metabolic stability 120 100
Stable compound
80 60 40
Metabolized compound
20 0 0
20
40
60
80
Time (min)
•MSIC in vitro assay •Allow determination of half life (min), elimination rate (1/min) and in vitro Clint (mL/min/kg) •Used to screen stable compound, PK prediction, etc.
Application #2 – Metabolic stability Human hepatocytes, 1e6 cells/mL, 37oC, 95/5% O2/CO2 120
HPLC-MS/MS LDTD-MS/MS
100
Bufuralol
%PR
80
60
40
Midazolam 20
0 0
20
40
60
80
100
120
Time (min)
Comparable results by HPLC-MS/MS and LDTD384-MS/MS
140
Application #2 – Metabolic stability Liver microsomes, 0.25mg/mL, 37oC, with NADPH N N
N
O
N O
N
Cl
O F
O
Phenacetin
Bufuralol
Midazolam LDTD384-Quantum Compound Midazolam
Bufuralol
Phenacetin
HPLC-4000QTRAP
species
Half Life (min)
Elimination Rate (1/min)
In Vitro Clint (mL/min/kg)
Half Life (min)
Elimination Rate (1/min)
In Vitro Clint (mL/min/kg)
human dog rat human dog rat human dog rat
8.0 2.0 2.2 79.4 26.0 3.7 65.9 38.0 105.0
0.0865 0.3443 0.3220 0.0087 0.0267 0.1852 0.0105 0.0182 0.0066
288 1983 >2000 29 154 >2000 35 105 48
4.2 2.6 1.7 106.9 25.9 1.7 80.2 24.2 65.4
0.1660 0.2673 0.4110 0.0065 0.0267 0.4084 0.0086 0.0286 0.0106
553 1540 >2000 22 154 >2000 29 165 76
Data generated by LDTD384-MS/MS are within 2 fold of the data generated by HPLC-MS/MS
Application #3 – Pharmacokinetics P.O.
5 mg/kg 0.5% Methocel t= 0h, 0.25h, 0.5h, 1h, 2h, 4h, 6h & 24h
Plasma quenched with 3 volumes ACN
Wistar Han Rats (n=3)
Application #3 – Pharmacokinetics WH rat PO @ 5mg/kg in 0.5% Methocel LDTD384-Quantum
RT: 0.22 - 2.01 SM: 7B
NL: 8.78E5 TIC F: + p APCI SRM ms2
[email protected] [143.010-143.110, 349.150-349.250] MS Quadran3_line_p
75
t=1h
70
t=0.5h
65
t=2h
60 55
Relative Abundance
50
•Less than 2 minutes to run all plasma samples from 1 rat
t=0.25h
45
t=4h
40
t=6h
35 30 25 20
t=8h
15 10 5
t=24h
t=0h
0 0.4
0.6
0.8
1.0
1.2 Time (min)
1.4
1.6
1.8
2.0
Application #3 – Pharmacokinetics WH rat dosed P.O. @ 5 mg/kg in 0.5% Methocel 10
HPLC-MS/MS LDTD-MS/MS
9
Concentration (uM)
8 7 6 5 4 3 2 1 0 0
5
10
15
20
Time (h)
Comparable results by HPLC-MS/MS and LDTD384-MS/MS However, the LOD is 3 fold higher by LDTD384-MS/MS (1.5nM vs 5nM)
25
LDTD384-MS/MS: Pros & Cons • Pros – – – –
Fast Æ 12 seconds sample-to-sample Autonomy of 3840 samples (10x 384well plates) Small amount of sample needed (1uL) Compatible with all mass spectrometer
• Cons – – – –
No separation Less compounds can be ionized vs ESI Some MRM with background LOD in plasma of 5nM (vs 1.5nM by HPLC-MS/MS) • Quantum vs API5000
Conclusion • LDTD384-MS/MS is: – 25x faster per sample than conventional HPLC-MS/MS methods – requiring a change in the way that the samples are treated vs HPLC-MS/MS • A challenge in a discovery environment with different compounds daily
– recommended for high throughput analysis of targeted compound (CYP inhibition for example)
Questions
Back-up
Ki/Kinact • Ki/Kinact Î Test compound at 0, 5, 10, 25 & 50uM Î Pre-incubation: 0’, 5’, 10’, 15’, 20’ Mifepristone 0.1
Ki = 50% Kinact
Maximal Kinact
0.08
Kobs (min-1)
0.06 y = m1*m0/(m2+m0) Error 0.0012171 0.16867
Chisq 5.1929e-06 R 0.99947
NA NA
m1 m2
0.04
0.02
Ki = inhibitor potency
Value 0.085791 1.3614
Compounds are ranked as low, moderate or high inhibitor based on Ki/Kinact
0
-0.02 -10
0
10
20
30
concentration (uM)
40
50
60
Biomek FX for optimal transfer to Lazwell plates
•Assay / samples preparation done in 96well format •Pool 4x 96well plates on 1x 384Lazwell •Transfer of 1uL are done using 10uL tips for optimal efficiency
Biomek FX for optimal transfer to Lazwell plates
•Sample pick-up •1uL accurately
•Sample delivery •1uL accurately •Sample can be delivered in selected Quadrant
Biomek FX for optimal transfer to Lazwell plates QUADRANT 1
QUADRANT 2
QUADRANT 3
QUADRANT 4
