BMS Propietary Information Karl-Heinz Ott 1
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Metabolomics: The quantitative analysis of endogenous metabolites in
biological samples
Rat, Mouse, Dog,Human, Cell lines
NMR, LC-MS, GC-MS of urine, serum, feces, tissues,
cell extracts,…
Screening for tox, efficacy, compliance & health Biomarkers
Physiology
Toxicity
MechanismMinimally invasive, preclinical and clinical evaluation of biofluids
Monitor predisposition, onset, duration, severity, and recovery in individuals
Simultaneous detection of multitude of metabolites
Comprehensive efficacy or tox screening Novel biomarkers identification
Insight into biochemical and physiological processes & mechanism-of-action
BMS Propietary Information Karl-Heinz Ott 2
Toxin-induced Cholestasis
Integrated Metabonomics and Transcriptomics for Hypothesis Generation and Mechanistic Studies
Advanvces in Metabolic Profiling,
Boston, Nov 13-15
Karl-Heinz Ott, Nelly Aranibar, Petia Shipkova, Serhiy Hnatyshyn, Mark Sanders,Steven Stryker , Brian Gemzik,Evan Janovitz,Bo Wen, Christine Huang,Wenjun Wang, Aiqing He
Bristol-Myers SquibbNov 2007
BMS Propietary Information Karl-Heinz Ott 3
Histopathology of bile duct @ day 10
Toxin 1 30 mg/kg/day x 10 days
Serum Bilirubin = 0.19 mg/dL
Compound 2 30 mg/kg/day x 10 days
Serum Bilirubin < 0.10 mg/dL
Inflammatory cell infiltrate
Plug frombile duct
Normal bile duct
Eroded bile duct epithelium
FibroplasiaHyperplastic bile duct epithelium
E. Janovitz
Drug effect on bile duct ephithelium?
BMS Propietary Information Karl-Heinz Ott 4
Study Design
P 1 3Control
P 1 310mpk
P 1 330mpk
168 24 168 24 168 24
LC
-MS For Bile Acids
Screening
Amino Acids
CPMG
2D-JRES
NOEGP
DeproteinizeNOEGP
NM
RL
C-M
S For Bile Acids
Screening
Amino Acids
NMR
LC/MS
NMR
Serum, and urine metabolomics evaluated by NMR and MS and Liver tissue expression profiling on 5 animals/group.Bile metabolom analyzed in second batch with 4 animals/groupExposure in plasma, tissue and bile (PK), histopathology and clinical chemistry endpoints evaluated for both batches.
Transcriptomics
BMS Propietary Information Karl-Heinz Ott 5
Taurocholate
Cholate
Glycocholate
Deoxycholate
Serum Bile Acids in Rats Treated with Toxin 1
10 mpk/Day1
10 mpk/Day3
30 mpk/Day1
30 mpk/Day3
Steven Stryker, Brian Gemzik (DT), Petia Shipkova, Nelly Aranibar, Mark Sanders (BDAS) and Karl-Heinz Ott (AG)
Cholic acid increased at day 1 and day 3 at 10mg/kg and at 30 mg/kg.
Cholate
10 mpk/Day1
10 mpk/Day3
30 mpk/Day1
30 mpk/Day3
10 mpk/Day1
10 mpk/Day3
30 mpk/Day1
30 mpk/Day3
Taurocholate
Strong increase in taurocholic acids at day 3
Taurocholate
Cholate
Glycocholate
Deoxycholate
BMS Propietary Information Karl-Heinz Ott 6
Timing of marker changes and spectrum of serum bile acid changes indicate that bile acid biosynthesis is not likely mechanistic cause of observed cholestasis
BMS Propietary Information Karl-Heinz Ott 7
NMR: Lipid and lipoprotein serum profile changes from LDL to HDL are highly significant upon treatment:
-CH2 -CH3
Red: High DoseBlue: Control
Source: Liposcience
BMS Propietary Information Karl-Heinz Ott 8
NMR Aromatic Region: aromatic amino acids response in intact serum
BMS Propietary Information Karl-Heinz Ott 9
TSP internal standard shift and broadening: a measure of hydrophobic protein binding
Binding, availability, and transport of hydrophobic molecules in the serum are altered, probably due to high bile acid concentrations on serum.
Red trace: high dose 3-day, TSP is less bound to protein and/or in fast exchange between free and bound states Blue trace: control, two protein-bound TSP populations in slow exchange with one another
BMS Propietary Information Karl-Heinz Ott 10
Urine Metabolom
Nelly Aranibar (BDAS) and Karl-Heinz Ott (AG)
Control 10mpk 30 mpk
Time Time Time
Sig
natu
re P
rofi
le V
alue
•Time-resolved PLS analysis indicated a dose response curve of the metabolite profiles. The magnitude of the metabolite changes underlying the profiles were considered to small to serve as a robust screening tools for a discovery program.
BMS Propietary Information Karl-Heinz Ott 11
PCA of Urine NMR data
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tPS
[2]
tPS[1]
DT07020U.070503.simca.M1 (PCA-X), PS-DT07020U.070503.simcatPS[Comp. 1]/tPS[Comp. 2]Colored according to Obs ID (Time_Day)
R2X[1] = 0.228632 R2X[2] = 0.129565 Ellipse: Hotelling T2PS (0.95)
008_0008_1008_3016_0016_1016_3024_0024_1024_3
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SIMCA-P+ 11.5 - 5/7/2007 12:55:14 PMAnimal 3101
•Animal 3101 has altered metabolism (starting pre-dose) (along PC2)•#3101, 3103, 3105 have more pronounced alteration than 3102, 3104•“Day” and “Time” have an effect that is similar in nature (i.e. along PC1) to possible “Dose” effect
BMS Propietary Information Karl-Heinz Ott 12
Plasma exposure data in Study 1
• Low exposure in Low dose
• High, but individually different exposure in High dose group
• 3103 and 3104 have lower exposure
• 3101 has altered metabolism, consistent with data from predose and day 1 urine metabolomics
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BMS Propietary Information Karl-Heinz Ott 13
Analysis of all metabolomics data in the context of exposure dataHierarchical Clustering
1.24 0
1 615 2 5 35
X3.0751.44X0.7C1.55X1.125C7.36X0.6653.845C7.415C8.365C0.64C8.12C8.175C8.57X7.5452.38C3.155X4.47X1.035.43X3.22.55X7.61X5.18C6.53C6.62X8.245C1.13X7.93X9.5X8.725C9.28C9.505C2.625X8.37.08C9.325X1.15X8.21C1.348.2257.45X6.375C9.745C9.61X5.9052.413.2756.83X0.725X3.369.9252.949.678.698.475X2.4C3.94
DgN DayN bms-772486 bms-736875 bms-800754
Clu
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ed li
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600
U &
S
Met
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Mea
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s
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Drug Meta-bolitesD
ayRed: Pos. correlated, green: negative correlated, black: uncorrelated
BMS Propietary Information Karl-Heinz Ott 14
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Var ID (Primary)
MS-Jres-cpmg-U.M2 (PCA-X), PCA Combined Mean/MeanStdp[Comp. 1]
R2X[1] = 0.234611 SIMCA-P+ 11.5 - 6/6/2007 4:51:09 PM
Serum and urine metabolites correlate closely with exposure
BMS Propietary Information Karl-Heinz Ott 15
Day
Animal - 1101
Animal - 2101
Animal - 3101
Animal - 1102
Animal - 2102
Animal - 3102
Animal - 1103
Animal - 2103
Animal - 3103
Animal - 1104
Animal - 2104
Animal - 3104
Animal - 1105
Animal - 2105
Animal - 3105
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High doseControl
6.585 7.58E-11 0 -11.3236 0.284948 0.391681 0.106733 16.3771 2.4264 3.51369 2.03924 5.39944
6.53 1.27E-07 0 -7.28839 0.360635 0.452251 0.091616 10.5095 4.5786 2.18343 7.07968 2.91848
6.49 6.09E-10 0 -11.3279 0.264445 0.373274 0.108829 18.023 6.9035 5.7871 2.61769 10.0591
ppm p-valueBootstrap t Mean(0)
Mean(3_3) Abs(diff)
Logp-value(Day)
Logp-value(T)
Logp-value(DG)
Logp-value(A(DG))
Logp-value(Day * DG)
A highly significant urinary biomarker is associated with treatment, time and toxicity
BMS Propietary Information Karl-Heinz Ott 16
TR-
control
HD, d3
Cannalicular transporters implied?
Three highly significant urinary markers match a metabolite previously discovered and unique to TR- rats urine
– TR- is a spontaneous mutation that impairs the cannalicular multispecific organic anion transporter (an ABC transporter aka. Multidrug-resistance associated protein, Mrp-2).
– Mrp2 transports [xenobiotica-] glucuronides, bilirubin (TR- are hyperbilirubinemic), and GSH complexes into bile
BMS Propietary Information Karl-Heinz Ott 17
NMR metabonomics analysis of Bile
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R.global
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R.local
110111011101110111031103
11031103
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21012101
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2101
K:\DT07020\B\amix/Mean00/1r
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K:\DT07020\B\amix/Mean08/1r
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K:\DT07020\B\amix/Mean24/1r
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K:\DT07020\B\amix/Mean24/1r
10.00 9.00 8.00 7.00 6.00
• 6h-8h bile composition differs most prominently from other time intervals, regardless of treatmentAnimal 1103 (injured) and 2101 (exposure/drug metabolism?) reflected as outlier
Control bile composition:22h-24h Predose differs from matching day1 and day3 collections.Only small differences between 2h-4h and 22h-24h
Local Pearson Correlation
BMS Propietary Information Karl-Heinz Ott 18
Average bile flow changes during 24h intervals-1
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Flo
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Red: ControlBlack: 30mpkN=4 for all control intervalsMean Flow = 2.0 ml/2hMean Flow at 8h high dose: 1.1 ml/2hMean Flow at 8h vehicle: 1.7 ml/2h
J. Lipid Res. Weis and Barth 19 (7): 856
Animals adapted to a restricted feeding regimen showed a significant increase of bile flow and of biliary bile salt and cholesterol excretion during feeding (10AM-3 PM); these parameters reached their maximum 3 hours after onset of food intake.
BMS Propietary Information Karl-Heinz Ott 19
Bile at different time periods, std. scaling
K:\DT07020\B\amix/Mean24/1r
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K:\DT07020\B\amix/Mean08/1rK:\DT07020\B\amix/Mean24/1r
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K:\DT07020\B\amix/Mean08/1r
Blue : 6h-8hBlack : 22h-24h
• Relative amount of signals in upfield and downfield region differs.
• Signature of bile acids is altered between 6-8h and 22-24h.
Spectra scaled to total integral (~ integral over 4—1ppm)
BMS Propietary Information Karl-Heinz Ott 20
K:\DT07020\B\amix/Mean08/1r
4.00 3.00 2.00 1.00
K:\DT07020\B\amix/Mean24/1rK:\DT07020\B\amix/Mean08/1r
9.00 8.00 7.00 6.00
K:\DT07020\B\amix/Mean24/1r
Black: 6h-8hBlue: 22h-24h
Upfield region
Downfield region
TS
P(8h) ~
TS
P(24h)
Spectra scaled to left portion (10-6ppm)
Bile at different time periods (rescaled)• Decrease of bile acids at 6-8h vs 22-24h. • Relative little change in aromatic portion between time periods.
BMS Propietary Information Karl-Heinz Ott 21
Control Day1,Day3
High dose Day1,Day3
Aliphatic (bile acid signal) region at 6h-8hMinimal changes upon treatment
Some alteration in bile acid composition
BMS Propietary Information Karl-Heinz Ott 22
Aromatic region at 6-8h shows marked differences upon treatment
controls
High dose day 3
High dose day 1
BMS Propietary Information Karl-Heinz Ott 23
Changes in aromatic region are observable immediately: 2-4h after
dose, day 1Control day 1
High dose day 3
High dose day 1
Control day 3
BMS Propietary Information Karl-Heinz Ott 24
Marker associated to Mrp2 transporter disappears from bile at high dose treatment•Marker appeared in urine spectra after treatment, but not in controls•Marker is “recovering” in animal 2101 at Day3 24h
Control day 1
High dose day 3
High dose day 1
Control day 3
22h-24h NMR spectra are shown
BMS Propietary Information Karl-Heinz Ott 25
Liver Expression ProfilingThe analysis of the expression data focused on CYPs and hepatocyte transporters that indicated:
• MRP3: Highly up-regulated upon treatment at day 3 (T1) and day 7 (T2)
• Basolateral Transporters: Sodium/bile acid co-transporter and other solute carrier anion transporters are predominantly down-regulated. Several other ABC transporters also downregulated.
• Cannalicular Transporters: MRP2 slightly upregulated; MRP1: downregulated; BSEP: ~ Constant.
• Pathway analysis did not show significant pathway associations except for pathways involving CYPs. CYPs: 3 up, 14 down (in 3day study),
• Cholesterol biosynthesis pathways are affected via altered CYP expression, but Cyp expression often different in T1/3day and T2/7day studies.
• (Non-CYP) genes involved in regulating bile production and transport were only sporadically found to be significantly altered.
• Inflammatory pathways were not indicated as affected.
Karl-Heinz Ott, Wenjun Wang, Aiqing He (AG) and Nelly Aranibar (BDAS)
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Fold Change Treated vs Control for selected transporters