2009 약물대사기반 심포지엄-정호상(3)

Evaluation of Drug Toxicity by Metabolom Profiling

Ho-Sang Jeong, Ph. D

Div. of Pharmacological Research, NIFDS

protein-gene interactions

protein-protein interactions

PROTEOME

GENOME

Citrate Cycle

METABOLISM

Bio-chemical reactions

From Genes To Metabolites

Metabolomics: A Multidisciplinary Study

Raw NMR spectraNMR spectra processed PCA

control

disease ortoxicity

STOCSY

UrineSerumtissue

Discovery of biomarkersIdentification of metabolic pathway

Year Budget (KWon) Contents

2006 32,000

Metabolomics를이용한내분비장애추정물질의안전성 예측 방법 연구(metabolic study of putative endocrine disruptor)

식욕억제제의약물동태 및 대사 연구(Pharmacokinetic study of anorexic drug using metabolomics)

2007 46,000

Metabolomics를이용한 간독성 평가방법 개발 (Liver toxicity study using metabolomics)

NSAIDs 약물의 약물이상반응에 대한 안전성 예측 연구 (1) (ADR study of NSAIDs using metabolomics)

대사체 생체지표를 이용한 내분비계 장애작용 예측 연구(metabolic study of putative endocrine disruptor)

And etc

2008 41,500

Metabolomics를이용한 신독성 평가방법 개발 (Kidney toxicity study using metabolomics)

NSAIDs 약물의 약물이상반응에 대한 안전성 예측 연구 (2) (ADR study of NSAIDs using metabolomics)

And etc

2009 37,000

간독성평가를위한 시험관내시험계에서의전사체대사체 발현 분석 연구(In vitro liver toxicity study using integrated omics technologies)

NSAIDs 약물의 약물이상반응에 대한 안전성 예측 연구 (3)(ADR study of NSAIDs using metabolomics)

And etc.

Total 156,500

Effort of NIFDS for Metabolomic Study

Metabolomics in Drug Toxicity

Metabolomic detection of liver and kidney toxicity

-30

-20

-10

0

10

20

30

-70 -60 -50 -40 -30 -20 -10 0 10 20 30 40 50 60 70

t[2]

t[1]

PLS-DA Class 1Class 2

SIMCA-P+ 11 - 13/03/2007 15:45:11

toxicity

control

Clinical Chemistry

Biomarkers for hepatotoxicity

NMR Spectrometry

Histopathology

Liver damage

D-GalactosamineSD

Hepatotoxicity

Acetaminophen

CCl4

blood

liver

urine

Multi-variate analysis

Study Design: Liver Toxicity

Treatment and sample collection: Liver Toxicity

CCl4 1 ml/kg, p.o

D-Galactosamine (GalN) 0.8 g/kg, i.p

Acetaminophen (AAP) 2 g/kg, p.o for 2 days

0 1 2

Day

Plasma clinical chemistry (blood)Histopathology (liver)

Urinal NMR (urine)

Study Design: Kidney Toxicity

Clinical Chemistry

Nephrotoxicity biomarkerMetabolomics

- Urine

Histopathology

Kidney Damage

GentamicinSD

Nephrotoxicity

Cis-platin

HgCl2

Administration HgCl2 0.1 or 0.75 mg/kg, ip

Gentamicin (GEN) 5 or 160 mg/kg for 2 days, ip

Cis-platin (CP) 5 mg/kg for 2 days or 20 mg/kg for single, ip

Kidney weight

p-Aminophenol

p-Aminophenol (PAP) 10 or 200 mg/kg, ip

Plasma clinical chemistry (blood)Histopathology (kidney)

Days

-1 0 1 2 3 4 5 6

Urinal NMR (urine)

Kidney wtUrinal clinical chemistry (urine)

Treatment

Biomaterial collection for HgCl2 treatment

Biomaterial collection for PAP treatment

0 1 2

Days

Treatment

3

Plasma clinical chemistry (blood)Histopathology (kidney)

Urinal NMR (urine)

Kidney wtUrinal clinical chemistry (urine)

Pattern recognition methodsSupervised techniques (PCA)

Unsupervised techniques (PLS-DA)

NMR

Import techniques and procedures in metabonomics

Control, Day 2 CCl4 1 ml/kg, Day 1 CCl4 1 ml/kg, Day 2

AAP 2 g/kg, Day 2 GalN 0.8 g/kg, Day 1

Con Day

1

Con Day

2 D

ay 1

4CCl D

ay 2

4CCl

Con Day

2

AAP Day

2

Con Day

1

Con Day

2

GalN D

ay 1

GalN D

ay 2

0

2

4

6

8

10

****

**

** **

CCl4 AAP GalNLi

ver

inju

ry s

core

A B C

ED F

Histopathology: Liver

NMR spectral analysis

1234567ppm

Target profiling Spectral binning

1234567ppm

hippurate urea

allantoincreatininehippurate

2-oxoglutarate

citrate

TMAO

succinatefumaratewater

creatinine

taurine

SIMCA-P ver.11 (Umetrics, Umeå, Sweden)

Partial Least Squares (PLS) discriminant analysis

Very importance variables (VIP) were also utilized to select putative markers for hepatotoxicity induced by CCl4, AAP, and GalN.

Multivariate statistical data analysis

Global profiling : Liver Toxicity

CCl4 AAP GalN

■ Day 0● Day 1♦ Day 2

A B C

Global profiling of hepatotoxicants

Class 1Class 5Class 9Class 11Class 16Class 19

GalN Day 1GalN Day 2CCl4 Day 1CCl4 Day 2AAP Day 2Control (Day 0)

Targeted profiling of CCl4

CCl4 Day 1CCl4 Day 2Con Day 0Con Day 1Con Day 2

A

B

Targeted profiling of AAP

AAP Day 1AAP Day 2Con Day 0Con Day 1Con Day 2

A

B

C

Targeted profiling of GalN

Con Day 0Con Day 1Con Day 2GalN Day 1GalN Day 2

A

B

2-Oxoglutarate

Con_CCl4_Day 1

CCl4_Day 1

Con_CCl4_Day 2

CCl4_Day 2

Con_AAP_Day 1

AAP_Day 1

Con_GalN_Day 1

GalN_Day 1

Con_GalN_Day 2

GalN_Day 20.0

2.5

5.0

7.5

10.0

**

**

*

Citrate

Con_CCl4_Day 1

CCl4_Day 1

Con_CCl4_Day 2

CCl4_Day 2

Con_AAP_Day 1

AAP_Day 1

Con_GalN_Day 1

GalN_Day 1

Con_GalN_Day 2

GalN_Day 20

1

2

3

4

5

** *

Taurine

Con_CCl4_Day 1

CCl4_Day 1

Con_CCl4_Day 2

CCl4_Day 2

Con_AAP_Day 1

AAP_Day 1

Con_GalN_Day 1

GalN_Day 1

Con_GalN_Day 2

GalN_Day 20

1

2

3

** *** *

Succinate

Con_CCl4_Day 1

CCl4_Day 1

Con_CCl4_Day 2

CCl4_Day 2

Con_AAP_Day 1

AAP_Day 1

Con_GalN_Day 1

GalN_Day 1

Con_GalN_Day 2

GalN_Day 20

1

2

3

4

*

Determination of endogenous metabolites

Acetate

Con_CCl4_Day 1

CCl4_Day 1

Con_CCl4_Day 2

CCl4_Day 2

Con_AAP_Day 1

AAP_Day 1

Con_GalN_Day 1

GalN_Day 1

Con_GalN_Day 2

GalN_Day 20

5

10

15

20

**

Betaine

Con_CCl4_Day 1

CCl4_Day 1

Con_CCl4_Day 2

CCl4_Day 2

Con_AAP_Day 1

AAP_Day 1

Con_GalN_Day 1

GalN_Day 1

Con_GalN_Day 2

GalN_Day 20.0

0.1

0.2

0.3

Lactate

Con_CCl4_Day 1

CCl4_Day 1

Con_CCl4_Day 2

CCl4_Day 2

Con_AAP_Day 1

AAP_Day 1

Con_GalN_Day 1

GalN_Day 1

Con_GalN_Day 2

GalN_day 20

2

4

6

8

10*

Allantoin

Con_CCl4_Day 1

CCl4_Day 1

Con_CCl4_Day 2

CCl4_Day 2

Con_AAP_Day 1

AAP_Day 1

Con_GalN_Day 1

GalN_Day 1

Con_GalN_Day 2

GalN_Day 20

1

2

3

4

5

**


Phenylacetate

Con_CCl4_Day 1

CCl4_Day 1

Con_CCl4_Day 2

CCl4_Day 2

Con_AAP_Day 1

AAP_Day 1

Con_GalN_Day 1

GalN_Day 1

Con_GalN_Day 2

GalN_Day 20.0

0.1

0.2

0.3

0.4

***

1-Methylnicotinamide

Con_CCl4_Day 1

CCl4_Day 1

Con_CCl4_Day 2

CCl4_Day 2

Con_AAP_Day 1

AAP_Day 1

Con_GalN_Day 1

GalN_Day 1

Con_GalN_Day 2

GalN_Day 20.0

0.2

0.4

0.6

*

*

Hippurate

Con_CCl4_Day 1

CCl4_Day 1

Con_CCl4_Day 2

CCl4_Day 2

Con_AAP_Day 1

AAP_Day 1

Con_GalN_Day 1

GalN_Day 1

Con_GalN_Day 2

GalN_Day 20.00

0.05

0.10

0.15

0.20

**

** *

Benzoate

Con_CCl4_Day 1

CCl4_Day 1

Con_CCl4_Day 2

CCl4_Day 2

Con_AAP_Day 1

AAP_Day 1

Con_GalN_Day 1

GalN_Day 1

Con_GalN_Day 2

GalN_4Day 20

1

2

3

**

**

*


-3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.5 1.0 1.5 2.0 2.5 3.0

Standard deviations from mean compound concentration

Compounds

Sam

ples

Hep

atot

oxic

ant

Con

trol

sFingerprint of endogenous metabolites

Results and conclusion

□ Current study provides that urinary 1H NMR spectral based metabolomics is a new approach to determine the change in endogenous metabolites in animals exposed to hepatotoxicants of CCl4, AAP, and GalN. NMR spectra showed pattern recognition using PLS-DA through distinct separation of clustering in hepatotoxicants-treated urine samples.

This pattern recognition may be used to screen hepatotoxic new drug candidates in early preclinical studies.

We proposed 12 putative biomarkers to predict hepatotoxicity induced by chemicals using NMR targeted analysis.

□ Classical clinical chemistry and histopathology provided validation for this study and this database of biomarker patterns may be useful for further study.

□ The suggested biomarkers should be explained how to involve in mechanism of hepatic injury. The further study with other hepatotoxicants needs to identify the endogenous biomarkers.

Histopathology : Kidney-HgCl2

×100

Control

HgCl2_0.75 mg/kg_D6

HgCl2_0.1 mg/kg_D6

HgCl2_0.75 mg/kg_D3

HgCl2_0.1 mg/kg_D3a b c

d e

Global Profiling (PCA): HgCl2

Global Profiling (PLS-DA): HgCl2

Targeted Profiling (PCA): HgCl2

Targeted Profiling (PLS-DA): HgCl2

Summary- HgCl2

Global profiling- clustering of samples by HgCl2 treatment - clear temporal profiling by HgCl2 administration

Target Profiling- similar to Global profiling- endogenous metabolites related to HgCl2 caused kindey toxicity: Glucose, acetate, allantoin, 2-oxoglutarate, alanine, lactate, formate, citrate, succinate, taurine, ethanol, hippurate

Histopathology : Kidney-Cisplatin

Control Cisplatin_5 mg/kg_D8

Cisplatin_20 mg/kg_D2



a

ed

cb

Global Profiling (PCA) : Cisplatin

Global Profiling (PLS-DA) : Cisplatin

Targeted Profiling (PLS-DA) : Cisplatin

Summary-Cisplatin

Global profiling - Clustering of samples by Cisplatin- Clear temporal profiling by Cisplatin

Target Profiling_PLS-CA- similar to global profiling- endogenous metabolites related to cisplatine caused kidney toxicity: N-acetylglycine, cinnamate, dimethylamine, hippurate, niacinamide, 2-oxoglutarate, lactate, cis-aconitine, 3-indoxylate, propionate, nicotinate, creatine, acetate, ethanol, phenylacetate

Histopathology : Kidney-Gentamicin

Control Gentamicin_5 mg/kg_D8

Gentamicin_160 mg/kg_D2



a

ed

cb

Global Profiling (PCA) : Gentamicin

Global Profiling (PLS-DA) : Gentamicin

Targeted Profiling (PLS-DA) : Gentamicin

Summary-Gentamicin

Global profiling - Clustering of samples by gentamicin- Clear temporal profiling by gentamicin

Target Profiling_PLS-DA- Similar to global profiling- Endogenous metabolites related to cisplatine caused kidney toxicity: Acetate, 2-oxoglutarate, glucose, allantoin, lactate, formate, citrate, glycine, taurine, benzoate, succinate, alanine

Histopathology : Kidney-p-Aminophenol

Control PAP_10 mg/kg_D3

PAP_200 mg/kg_D1

PAP_10 mg/kg_D1

PAP_200 mg/kg_D3

a

ed

cb

Global Profiling (PCA) : p-Aminophenol

Global Profiling (PLS-DA) : p-Aminophenol

Targeted Profiling (PCA) : p-Aminophenol

Targeted Profiling (PLS-DA) : p-Aminophenol

Summary: p-Aminophenol

Global profiling - clustering of smaples by p-aminophenol- Stable temporal profiling by p-aminophenol

Target Profiling_PLS-DA- Similar to global profiling- Endogenous metabolites related to p-aminophenolcaused kidney toxicity: Glucose, lactate, alanine, 2-oxoglutarate, acetate, allantoin, citrate, formate, taurine, creatine, succinate

Common Metabolic Markers

HgCl2Cisplatin

Gentamicin p-Aminophenol

glucoseacetateallantoin

2-oxoglutaratealanine

lactate

formate

citrate

succinate

taurine

ethanol

hippurate

N-acetylglycinecinnamate

dimethylamineniacinamide

Cis-aconitine3-indoxylate

propionatenicotinate

creatine

phenylacetate

glycine

benzoate

Determination of endogenous metabolites (2-oxoglutarate)

HgCl2

D0 D2 D3 D4 D5 D6 D0 D2 D3 D4 D5 D60

2

4

6

8

10Hg_0.1Hg_0.75

**

Days after treatment

2-O

xogl

utar

ate

Cisplatin

D0 D2 D3 D4 D5 D6 D7 D8 D0 D2 D3 D4 D5 D6 D7 D80

1

2

3

4

5CP_20CP_5

***

**

**

** ** ** ** **


2-O

xogl

utar

ate

Gentamicin


2

4

6GEN_5GEN_160

*

** ** ** ** **


2-O

xogl

utar

ate

p-Aminophenol

D0 D2 D3 D0 D2 D30

1

2

3PAP_10PAP_200

**

**

**


2-O

xogl

utar

ate

Determination of endogenous metabolites (Taurine)

HgCl2

D0 D2 D3 D4 D5 D6 D0 D2 D3 D4 D5 D60.0

0.5

1.0

1.5Hg_0.1Hg_0.75

**

**** ** **


Taur

ine

Cisplatin


1

2

3

4

5CP_20CP_5

**


Taur

ine

Gentamicin

D0 D2 D3 D4 D5 D6 D7 D8 D0 D2 D3 D4 D5 D6 D7 D80.0

0.5

1.0

1.5

2.0

2.5GEN_5GEN_160


Taur

ine

p-Aminophenol

D0 D2 D3 D0 D2 D30.0

0.5

1.0

1.5PAP_10PAP_200

**


Taur

ine

-3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.5 1.0 1.5 2.0 2.5 3.0

Standard deviations from mean compound concentrationEn

doge

nous

met

abol

ites

NephrotoxicantsControl

Fingerprint of endogenous metabolites

Metabolic markers for Nephrotoxicity and Hepatotoxicity

Biomarkers Nephrotoxicity(2008)

Hepatotoxicity(2007)

acetatelactate

alanine

allantoincitrate

2-oxoglutarate

glucose -formate -

taurine

succinatephenylacethyl glycine -

1-methylnicotinamide -

hippurate -

benzoate -phenylacetate -

2009 약물대사기반 심포지엄-정호상(3)

Education