challenges with nucleoside triphosphate method development
TRANSCRIPT
Challenges with nucleoside triphosphate method development and analysis during
the life cycle of a HCV program
Janssen Research & Development
Drug Safety Sciences/ Bioanalysis
Liesbeth Vereyken
1
Outline of the presentation
• Background – situation of project
• Method development for nucleoside triphosphate quantitation ▫ Challenges ▫ Extraction recovery ▫ Stability ▫ LCMSMS
• Application of method
▫ In vivo ▫ In vitro support
• Method development for endogenous triphosphates
• Conclusion
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• Up to 3% of population infected (170 million worldwide) • 80% will remain chronically infected for decades • Nucleoside drugs under development for treatment of HCV
Background Hepatitis C Virus (HCV)
Passive
diffusion PepTr Nucleoside
Kinase Nucleotidyl Kinase
Nucleoside Diphosphate Kinase
Incorporation In DNA
Nuc Tr
Antiviral effect
Nucleoside drug Nuc MP DP TP
Liver cell
MP = monophosphate DP = diphosphate TP = triphosphate
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Method development for TP quantitation
• Request for triphosphate (TP) analysis in pre-NME toxicity study in liver
• Literature: LC methods ▫ Ion-pairing: non-volatile solvent ▫ Ion-exchange: pH gradient, conc buffer
gradient
MS methods ▫ Negative mode: phosphate groups, more
sensitivity ▫ Positive mode: base, better selectivity
Lit ref: J.Sep.Sci. 2009, 32, 1275-1283
• Challenges:
▫ Extraction
▫ Sample handling
▫ Stability
▫ Robust chromatography
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Method development for TP quantitation Challenges
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Method development for TP quantitation Challenges
• Standard protocol for tissue homogenisation and extraction ▫ 1/10 w/w homogenisation in aqueous buffer
▫ Extraction with 3 volumes of organic (methanol)
• Stability issues anticipated:
▫ homogenates in MeOH/EDTA-EGTA
▫ inactivate phosphatase activity – solubility of TP
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Method development for TP quantitation Extraction recovery
MATRIX % recovery vs reference
with evaporation
without evaporation
methanol/water 70/30 (EDTA/EGTA 20 mM)
86 109
liver homogenate 37 42
• 100 ml homogenate (70/30 Methanol/20mM EDTA-EGTA (4°C))
• 100 ml TP in water
• Add 200 ml methanol and vortex
• centrifugate (9000g) - directly inject supernatant OR
• evaporate to dryness, redissolve in 200 ml 25mM NH4Ac/MeOH (70/30)
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Method development for TP quantitation Extraction recovery
• 100 ml homogenate (70/30 Methanol/20mM EDTA-EGTA (4°C))
• 100 ml TP solution in water
• Vortex
• NO ADDITION OF extra METHANOL
• centrifugate (9000g) - directly inject supernatant (SN)
% recovery
SN homogenate 83 87 95
Resuspension solvent water methanol methanol/water
70/30
first resuspension in 200 µl solvent
21 0.5 8
2nd resuspension in 200 µl solvent
3 0 3
total 107 87 102
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% of reference (t=0 min)
Incubation Condition
30 min 60 min 120 min
Liver homogenate
TP 4°C 100 99 98
RT 99 97 89
DP 4°C 100 103 112
RT 107 118 154
Supernatant
TP 4°C 108 109 108
RT 99 97 89
DP 4°C 102 103 105
RT 104 103 115
Method development for TP quantitation Benchtop stability in liver homogenate and SN
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Method development for TP quantitation LC-MS/MS
Column: Biobasic AX (Thermo) 50x4.6 mm, 5µm flow rate 0.8 ml/min A: 0.025M ammonium acetate pH 6.0 (adjusted with HCOOH) B: acetonitrile (constant @ 10%) C: 0.025M ammonium acetate pH 10.0 (adjusted with NH4OH 25%)
XIC of +MRM (4 pairs): 523.1/112.0 amu from Sample 1 (LSIP-01-031) of 031.wiff (Turbo Spray) Max. 4.6e4 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 Time, min
0.0
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
3.0e4
3.5e4
4.0e4
4.5e4
Intensity, cps
4.10
Printing Date: Friday, 14 November 2008 Printing Time: 13:53:53 Operator: Noels, Annemie [PRDBE]
*BAN 551-9
Results Path: N/A Method Path: D:\Studies\analyst data\Projects\JNJ-42088527-BA1032-01\Quantitation Methods Page 1 of 1
Analyst Version: 1.4.2
TP
DP
MP
parent
0
20
40
60
80
100
%
C
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• Ion exchange column ▫ variability in column batches – need for optimisation ▫ difference in retention time (Rt) ▫ adapt buffer concentration for similar retention times ▫ fresh preparation of 0.025M NH4Ac pH10 to avoid shift in Rt
• Other HPLC columns were evaluated (C18, C8, Hilic, Amide)
▫ no sensitive alternative method was found
• For some TP analogues a post-column make up flow with MeOH is needed to obtain good sensitivity
Method development for TP quantitation LC-MS/MS
• Quantitate NTP in liver in rat pre-NME tox study
▫ Liver sampled and stored @ -80 °C
▫ Homogenisation @ time of analysis in ice cold MeOH/EDTA-EGTA
▫ Batch acceptance criteria OK
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Application of method In vivo rat study
0
50
100
150
200
250
0 10 20 30
ng
/g
liv
er
time after administration (h)
Triphosphate levels after 5 days RD 200 mg/kg (ng/g)
• Repeat study
▫ Liver sampled and immediate homogenisation
▫ TPs detected
No TPs were detected in study samples
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Application of method Next steps
Questions: • Are TPs stable in solid tissues post sampling?
• Impact of homogenisation procedure on results
▫ whole liver versus biopts ▫ snap freezing (in liquid N2)
conc ng/g
% of reference
immediately homogenise liver in ice cooled MeOH/EGTA-EDTA
2405 100
snap-freeze liver in liquid N2, homogenise after 2h
613 26
cut liver into pieces, liquid N2, homogenise after 2h
246 10
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Application of method Dog liver biopts
• 20 mg biopts sampled under laparoscopy
▫ Snap frozen in liquid N2 at the time of sampling
▫ addition of 70% MeOH/20mM EDTA-EGTA
▫ Immediate homogenisation with ultrasonic probe
• Sample analysis within 24 h
• Results:
▫ Liver triphosphate levels (ng/g) after 7 d RD in dog 1150 ng/g @ 1h – 899 ng/g @ 6h following last dose
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Application of method In vitro radioactive studies
Rat hepatocytes – cpd A
JNJ-42088527; MeOH-EDTA-EGTA cellysaat monkey M Met Id
mV
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
55.00
60.00
65.00
70.00
75.00
80.00
85.00
90.00
95.00
100.00
105.00
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00
JNJ-42050034; MeOH-EDTA-EGTA monkey cellysaat monkey M Met. Id
mV
-20.00
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
160.00
180.00
200.00
220.00
240.00
260.00
280.00
300.00
320.00
340.00
360.00
380.00
400.00
420.00
440.00
460.00
480.00
500.00
520.00
540.00
560.00
580.00
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00
Monkey hepatocytes – cpd A
parent
parent
TP
TP
TP
TP
parent
parent
MP DP
Rat hepatocytes – cpd B Monkey hepatocytes – cpd B Rad a
bundance
Rad a
bundance
Rad a
bundance
Rad a
bundance
Time (min) Time (min)
Time (min) Time (min)
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Conclusion part 1
• Successful TP analysis depends on:
• Ion exchange LC
• MS/MS in positive ion mode
• After sampling, immediate homogenisation in 70/30 MeOH/ 20mM EDTA-EGTA
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Method development for endogenous TP quantitation
• Lead project: need for analysis of endogenous TP levels in tissues (liver and bone marrow) and in in vitro cell lines
• Analytes of interest: d-CTP, CTP and d-GTP
Deoxy- GTP (deoxyguanosine triphosphate)
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• Liver and cell samples were processed as optimized for exogenous TP analysis
• Bone marrow was extracted from femur under air pressure and immediately sonicated in ice cold MeOH/20mM EDTA-EGTA solution
• Stable isotope labelled IS (STIL) added to compensate for differences in response in different matrices
• LC adapted for separation between different NTP
Method development for endogenous TP quantitation
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column: Biobasic AX (Thermo) 50x4.6 mm, 5µm Flow 0.500 ml/min A: 0.01M ammonium acetate pH 5.4 (adjusted with CH3COOH) B: acetonitrile C: 0.01M ammonium acetate pH 10.7 (adjusted with NH4OH 25%)
MS parameters
Q1 Q3
d-CTP 468 112
STIL d-CTP 480 119
CTP 484 112
STIL CTP 496 119
d-GTP 508 152
STIL d-GTP 523 162
Method development for endogenous TP quantitation
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Application: Endogenous nucleoside triphosphate quantitation
0
10
20
30
40
50
60
70
80
90
100
%
C
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Application: Endogenous/exogenous nucleoside triphosphate quantitation
exogenous
nu
cle
os
ide
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Conclusion
• Endogenous TP analysis: • LC method with minor modifications for separation of
endogenous triphosphates
• MS/MS in positive ion mode – selective base ion in Q3
• Sampling method optimised per sample type (different tissues, in vitro samples)
Acknowledgements Lieve Dillen Philip Timmerman Luc Sips Ronald de Vries Laurent Leclercq Bas Jan Vanderleede
Janssen Research & Development
Drug Safety Sciences
Filip Cuyckens Nadine Pauwels Willy Lorreyne Sophie Lachau-Durand Iris Vanwelkenhuysen Marlies De Boeck Freddy Van Goethem