2009 약물대사기반 심포지엄-정호상(3)
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2009 약물대사기반 심포지엄-정호상(3)

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    2009 약물대사기반 심포지엄-정호상(3) 2009 약물대사기반 심포지엄-정호상(3) Presentation Transcript

    • Evaluation of Drug Toxicity by Metabolom Profiling Ho-Sang Jeong, Ph. D Div. of Pharmacological Research, NIFDS
    • From Genes To Metabolites GENOME protein-gene interactions PROTEOME protein-protein interactions METABOLISM Bio-chemical reactions Citrate Cycle
    • Metabolomics: A Multidisciplinary StudyUrineSerumtissue NMR spectra processed PCA Raw NMR spectra control STOCSY Discovery of biomarkers disease orIdentification of metabolic pathway toxicity
    • Effort of NIFDS for Metabolomic Study Year Budget (KWon) Contents Metabolomics를 이용한내분비장애추정물질의 안전성 예측 방법 연구 2006 32,000 (metabolic study of putative endocrine disruptor) 식욕억제제의 약물동태 및 대사 연구 (Pharmacokinetic study of anorexic drug using metabolomics) Metabolomics를 이용한 간독성 평가방법 개발 (Liver toxicity study using metabolomics) NSAIDs 약물의 약물이상반응에 대한 안전성 예측 연구 (1) (ADR study of NSAIDs using metabolomics) 2007 46,000 대사체 생체지표를 이용한 내분비계 장애작용 예측 연구 (metabolic study of putative endocrine disruptor) And etc Metabolomics를 이용한 신독성 평가방법 개발 (Kidney toxicity study using metabolomics) NSAIDs 약물의 약물이상반응에 대한 안전성 예측 연구 (2) (ADR study of NSAIDs using metabolomics) 2008 41,500 And etc 간독성평가를 위한 시험관내시험계에서의 전사체대사체 발현 분석 연구 (In vitro liver toxicity study using integrated omics technologies) 2009 37,000 NSAIDs 약물의 약물이상반응에 대한 안전성 예측 연구 (3) (ADR study of NSAIDs using metabolomics) And etc. Total 156,500
    • Metabolomics in Drug Toxicity
    • Metabolomic detection of liver and kidneytoxicity PLS-DA Class 1 Class 2 30 control 20 10 t[2] 0 -10 -20 toxicity -30 -70 -60 -50 -40 -30 -20 -10 0 10 20 30 40 50 60 70 t[1] SIMCA-P+ 11 - 13/03/2007 15:45:11
    • Study Design: Liver Toxicity Hepatotoxicity Clinical Chemistry Liver damage blood Histopathology CCl4 Acetaminophen liver SD D-Galactosamine urine NMR Spectrometry Multi-variate analysis Biomarkers for hepatotoxicity
    • Treatment and sample collection: Liver Toxicity CCl4 1 ml/kg, p.o Acetaminophen (AAP) 2 g/kg, p.o for 2 days D-Galactosamine (GalN) 0.8 g/kg, i.p Plasma clinical chemistry (blood) Histopathology (liver) Urinal NMR (urine) 0 1 2 Day
    • Study Design: Kidney Toxicity Nephrotoxicity Clinical Chemistry HgCl2 Kidney weight Kidney Damage Cis-platin SD Histopathology Gentamicin p-Aminophenol Metabolomics - Urine Nephrotoxicity biomarker Administration HgCl2 0.1 or 0.75 mg/kg, ip Cis-platin (CP) 5 mg/kg for 2 days or 20 mg/kg for single, ip Gentamicin (GEN) 5 or 160 mg/kg for 2 days, ip p-Aminophenol (PAP) 10 or 200 mg/kg, ip
    • Biomaterial collection for HgCl2treatment Plasma clinical chemistry (blood) Histopathology (kidney) Kidney wt Urinal clinical chemistry (urine) Urinal NMR (urine) Treatment -1 0 1 2 3 4 5 6 Days
    • Biomaterial collection for PAP treatment Plasma clinical chemistry (blood) Histopathology (kidney) Kidney wt Urinal clinical chemistry (urine) Urinal NMR (urine) Treatment 0 1 2 3 Days
    • Import techniques and procedures inmetabonomics NMR Pattern recognition methods Supervised techniques (PCA) Unsupervised techniques (PLS-DA)
    • Histopathology: Liver Control, Day 2 CCl4 1 ml/kg, Day 1 CCl4 1 ml/kg, Day 2A B C AAP 2 g/kg, Day 2 GalN 0.8 g/kg, Day 1 10 FD E 8 ** Liver injury score ** 6 ** ** 4 ** 2 0 1 2 2 1 2 1 2 2 1 2 ay ay ay ay ay ay ay ay ay ay D D D D D D D D D D N N on on on P on on l4 l4 A C C al al C C C C C A C C G G CCl4 AAP GalN
    • NMR spectral analysis ppm 7 6 5 4 3 2 1 Target profiling Spectral binning TMAO creatinine hippurate allantoincreatinine taurine citrate hippurate urea 2-oxoglutarate water fumarate succinate ppm 7 6 5 4 3 2 1
    • Multivariate statistical data analysis  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.
    • Global profiling : Liver Toxicity ■ Day 0 ● Day 1 ♦ Day 2 CCl4 AAP GalNA B C
    • Global profiling of hepatotoxicants GalN Day1 Class 1 GalN Day5 Class 2 CCl4 Day 1 Class 9 CCl4 Day 11 Class 2 AAP Day 16 Class 2 Control (Day 0) Class 19
    • Targeted profiling of CCl4 B A CCl4 Day 1 CCl4 Day 2 Con Day 0 Con Day 1 Con Day 2
    • Targeted profiling of AAP B AAP Day 1 A AAP Day 2 Con Day 0 Con Day 1 Con Day 2 C
    • Targeted profiling of GalN B A Con Day 0 Con Day 1 Con Day 2 GalN Day 1 GalN Day 2
    • C C on on _C _C C C l4 0.0 2.5 5.0 7.5 10.0 l4 _D 0 1 2 3 _D C ay C ay C C C C on l4_ 1 ** on l4_ 1 _ C Da C y * _ C Da C y l4 1 l4 1 _D _D C ay C ay C Cl4 2 2 on _ C Cl4 on _D _ A Da * A y ** _A ay P 2 A 2 _D P _D A ay C A A ay C A on P_ 1 on P_ 1 _ G Da y * Taurine _ G Da al y N 1 al 1 _D N G ay _D a C 2-Oxoglutarate G ay C a on lN_ 1 _ G Da ** on lN_ 1 y ** al _ G Da N 1 al y _D N 1 G _D ay al G ay N 2 al _D N 2 ay _D ay 2 2 CC on on _C _C C C l4 0 1 2 3 4 5 l4 _D 0 1 2 3 4 _D C ay C ay C CC C on l4_ 1 on l4_ 1 _ C Da ** _ C Da C y C y l4 1 l4 1 _D _D C ay C ay C Cl4 2C Cl4 2 on _ on _ * _ A Da A y * _ A Da A y P 2 P 2 _D _D A ay A ay C AC A on P_ 1 Citrate on P_ 1 _G D _G D ay ay al N 1 Succinate al N 1 _ _D G Da G ay C a yC a on lN_ 1 on lN_ 1 _G D _G D ay ay al al N 1 N 1 _D _ G ay G Da Determination of endogenous metabolites al al y N 2 N 2 _D _D ay ay 2 2
    • C C on on _C _C C C l4 0.0 0.1 0.2 0.3 l4 0 5 10 15 _D _D 20 C ay C C 1 C ay C l C l4 1 on 4_ D on _ ** _C ay _ C Da C 1 C y l4 l4 1 _D _D C ay C ay C 2 C C 2 C l4 on l4 _D on _D _A ay _A ay A 2 A 2 P P _D _D A ay A ay A C A C 1 on P_ 1 Acetate on P_ D _ G Da _G ay al y al 1 N 1 N _D _D G Betaine ay G ay C al N 1 C al N 1 on _ on _D _ G Da _G al y ay N 1 al N 1 _D _D G ay al 2 G ay N al N 2 _D _D ay ay 2 2 C onC _C on _C C l4 0 2 4 6 8 10 C _D l4 C _ 0 1 2 3 4 5 ay C C C DaC C y on l4_ 1 on l4 1 _ C Da _C _D C y l4 1 C ay _D l4 _ 1 C ay C Da C Cl4 2C Cl y on _ on 4_ 2 _ A Da _A D A y2 A ay P P _D _ 2 A a A Da C A yC on P_ 1 o n AP y 1 Lactate _ G Da _G _D al y 1 Allantoin al ay N N _D _ 1 G a G DaC a C alN y 1 on lN y 1 on _ _G _D _ G Da al y al ay N 1 N _D _ 1 a G Determination of endogenous metabolites G Da al y al y 2 N N _d * _D 2 ay ** ay 2 2
    • CC on on _C _C C C l4 0.0 0.1 0.2 0.3 l4 _D 0.4 0.00 0.05 0.10 0.15 0.20 _D C ay C ay C CC C on l4_ 1 on l4_ 1 _ C Da * _ C Da C y ** C y l4 1 l4 1 _D _D C ay C ay C Cl4 2C Cl4 2 on _ on _ _ A Da _ A Da A y A y P 2 P 2 _D _D A ay A ay C AC A on P_ 1 ** on P_ 1 _ G Da _ G Da y al y al N 1 N 1 Phenylacetate _D _D G ay Hippurate G ay C aC a on lN_ 1 on lN_ 1 _ G Da * _ G Da y al y al N 1 N 1 _D _D G ay G ay al al N 2 N 2 _D _D ay ay ** 2 2 C onC _C on C _C l4 0.0 0.2 0.4 0.6 C _D l4 0 1 2 3 C ay _D C 1 C ay C l4C C 1 on _D * on l4_ _C ay _ C Da C 1 y l4 C 1 _D l4 _D C ay C C 2 C ay C l4C l4 2 on _D on _D _A ay ** _A ay A 2 A P P 2 _D _D A ay A ay C A 1C A 1 on P on P_ _D _G ay _ G Da y al N 1 al N 1 _D _D G a G Benzoate ay al y 1C al 1 C N 1-Methylnicotinamide o n N_ on _ * _ G Da ** _ G Da y al y 1 al N 1 N _D _D G G a Determination of endogenous metabolites al ay N al y 2 N _4 2 _D D * ay ay 2 2
    • Fingerprint of endogenous metabolites Standard deviations from mean compound concentration -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.5 1.0 1.5 2.0 2.5 3.0 Compounds ControlsSamples Hepatotoxicant
    • 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 Control HgCl2_0.1 mg/kg_D3 HgCl2_0.1 mg/kg_D6 a b c d e ×100 HgCl2_0.75 mg/kg_D3 HgCl2_0.75 mg/kg_D6
    • Global Profiling (PCA): HgCl2
    • Global Profiling (PLS-DA): HgCl2
    • Targeted Profiling (PCA): HgCl2
    • Targeted Profiling (PLS-DA): 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_D2 Cisplatin_5 mg/kg_D8 a b c d e Cisplatin_20 mg/kg_D2 Cisplatin_20 mg/kg_D8
    • Global Profiling (PCA) : Cisplatin
    • Global Profiling (PLS-DA) : Cisplatin
    • Targeted 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_D2 Gentamicin_5 mg/kg_D8a b c d e Gentamicin_160 mg/kg_D2 Gentamicin_160 mg/kg_D8
    • Global Profiling (PCA) : Gentamicin
    • Global Profiling (PLS-DA) : Gentamicin
    • Targeted 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_D1 PAP_10 mg/kg_D3a b c d e PAP_200 mg/kg_D1 PAP_200 mg/kg_D3
    • Global Profiling (PCA) : p-Aminophenol
    • Global Profiling (PLS-DA) : p-Aminophenol
    • Targeted Profiling (PCA) : p-Aminophenol
    • Targeted Profiling (PLS-DA) : 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-aminophenol caused kidney toxicity: Glucose, lactate, alanine, 2- oxoglutarate, acetate, allantoin, citrate, formate, taurine, creatine, succinate
    • Common Metabolic Markers cinnamate N-acetylglycine Cisplatin HgCl2 dimethylamine niacinamide ethanol Cis-aconitine hippurate 3-indoxylate propionate nicotinate 2-oxoglutarate phenylacetate alanine citrate lactate allantoin succinate acetate glucose formate creatine glycine taurine benzoate p-AminophenolGentamicin
    • Determination of endogenous metabolites(2-oxoglutarate) HgCl2 Cisplatin 5 10 CP_20 Hg_0.1 CP_5 Hg_0.75 4 8 ** 2-Oxoglutarate 2-Oxoglutarate 6 3 4 2 ** 2 1 ** * ** ** 0 ** ** ** ** 0 D0 D2 D3 D4 D5 D6 D0 D2 D3 D4 D5 D6 D0 D2 D3 D4 D5 D6 D7 D8 D0 D2 D3 D4 D5 D6 D7 D8 Days after treatment Days after treatment Gentamicin p-Aminophenol 6 3 GEN_5 PAP_10 GEN_160 PAP_200 * 4 2 2-Oxoglutarate 2-Oxoglutarate 2 1 ** ** ** ** ** ** ** ** 0 0 D0 D2 D3 D4 D5 D6 D7 D8 D0 D2 D3 D4 D5 D6 D7 D8 D0 D2 D3 D0 D2 D3 Days after treatment Days after treatment
    • Determination of endogenousmetabolites (Taurine) HgCl2 Cisplatin 5 1.5 CP_20 Hg_0.1 CP_5 Hg_0.75 ** 4 1.0 3 Taurine Taurine 2 0.5 ** 1 ** ** ** ** 0.0 0 D0 D2 D3 D4 D5 D6 D0 D2 D3 D4 D5 D6 D0 D2 D3 D4 D5 D6 D7 D8 D0 D2 D3 D4 D5 D6 D7 D8 Days after treatment Days after treatment Gentamicin p-Aminophenol 2.5 1.5 GEN_5 PAP_10 GEN_160 PAP_200 2.0 1.0 1.5 Taurine Taurine 1.0 0.5 ** 0.5 0.0 0.0 D0 D2 D3 D4 D5 D6 D7 D8 D0 D2 D3 D4 D5 D6 D7 D8 D0 D2 D3 D0 D2 D3 Days after treatment Days after treatment
    • Fingerprint of endogenous metabolites Standard deviations from mean compound concentration -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.5 1.0 1.5 2.0 2.5 3.0 Control Nephrotoxicants Endogenous metabolites
    • Metabolic markers for Nephrotoxicity andHepatotoxicity Biomarkers Nephrotoxicity Hepatotoxicity (2008) (2007) acetate lactate alanine allantoin citrate 2-oxoglutarate glucose - formate - taurine succinate phenylacethyl glycine - 1-methylnicotinamide - hippurate - benzoate - phenylacetate -