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

2

• 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

3

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

4

Method development for TP quantitation Challenges

5

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

6

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)

7

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

8

% 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

9

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

10

• 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

11

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

12

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

13

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

14

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)

15

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

16

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)

17

• 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

18

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

19

Application: Endogenous nucleoside triphosphate quantitation

0

10

20

30

40

50

60

70

80

90

100

%

C

20

Application: Endogenous/exogenous nucleoside triphosphate quantitation

exogenous

nu

cle

os

ide

21

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