Metabolomics: The quantitative analysis of endogenous metabolites in biological samples

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


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


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?


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


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


Timing of marker changes and spectrum of serum bile acid changes indicate that bile acid biosynthesis is not likely mechanistic cause of observed cholestasis


NMR: Lipid and lipoprotein serum profile changes from LDL to HDL are highly significant upon treatment:

-CH2 -CH3

Red: High DoseBlue: Control

Source: Liposcience


NMR Aromatic Region: aromatic amino acids response in intact serum


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


Urine Metabolom

Nelly Aranibar (BDAS) and Karl-Heinz Ott (AG)

Control 10mpk 30 mpk

Time Time Time

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•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.


PCA of Urine NMR data

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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


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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Analysis of all metabolomics data in the context of exposure dataHierarchical Clustering

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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

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Drug Meta-bolitesD

ayRed: Pos. correlated, green: negative correlated, black: uncorrelated


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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


Day

Animal - 1101

Animal - 2101

Animal - 3101

Animal - 1102

Animal - 2102

Animal - 3102

Animal - 1103

Animal - 2103

Animal - 3103

Animal - 1104

Animal - 2104

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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


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


NMR metabonomics analysis of Bile

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K:\DT07020\B\amix/Mean00/1r

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• 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


Average bile flow changes during 24h intervals-1

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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.


Bile at different time periods, std. scaling


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K:\DT07020\B\amix/Mean08/1rK:\DT07020\B\amix/Mean24/1r

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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)



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Black: 6h-8hBlue: 22h-24h

Upfield region

Downfield region

TS

P(8h) ~

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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.


Control Day1,Day3

High dose Day1,Day3

Aliphatic (bile acid signal) region at 6h-8hMinimal changes upon treatment

Some alteration in bile acid composition


Aromatic region at 6-8h shows marked differences upon treatment

controls

High dose day 3

High dose day 1


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


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


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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Documents

Metabolomics: The quantitative analysis of endogenous metabolites in biological samples