Metabolomics Analysis Suggests Role of 3-Indoxyl Sulfate in Central Nervous System Toxicity and an Early Indicator of Chemically-Induced Renal FailureJoanna Zgoda-Polsa, Swapan Chowdhurya, Mark Wirtha, Kevin Altona, Danny Alexanderb, Michael Milburnb | aDMPK, Merck Research Laboratories, Kenilworth, NJ | bMetabolon, Inc., Durham, NCwww.metabolon.com | 919.572.1711

FoCus oN MetaboLIsMBiochemistry Advantages any sample type.

Many applications.

Fewer total biochemicals.

Solutions through Understanding Metabolism

GLobaL MetaboLoMICs teChNoLoGyMetabolomics is defined as “the non-biased quantification and identification of all metabolites present in a biological system” but in practice the term metabolomics is used in a rather broad sense and covers many different analytical methodologies. to address the challenges associated with metabolomics, a comprehensive, integrated analytical and data handling platform was developed that provides a chemo-centric global metabolomics analyses of biological systems. the analytical platform incorporates two separate uhPLC/Ms/Ms2 methods and a GC/Ms method which increases the overall coverage of small molecules in a biological sample. the resulting Ms and/or Ms2 data are searched against an in-house generated authentic standard library which includes retention time, molecular mass to charge (m/z), preferred adducts and in-source fragment information as well as the associated Ms fragmentation spectra for all biochemicals in the library. the library enables the rapid and accurate identification of the experimentally detected small molecules based on a multi-parameter match without need for time consuming further analysis. this integrated platform allows the robust, high-throughput collection and analysis of analytical data and accurately identifies a large number and broad spectrum of biochemicals thereby facilitating biochemical interpretation of metabolomic experiments.

Integrated, nontargeted ultrahigh performance liquid chromatography/electrospray ionization tandem mass

spectrometry platform for the identification and relative quantification of the small-molecule complement of biological systems. Evans AM, DeHaven CD, Barrett T, Mitchell M, Milgram E., Metabolon, Inc. Anal Chem. 2009 Aug 15;81(16):6656-67.

Organization of GC/MS and LC/MS metabolomics data into chemical libraries. Dehaven CD, Evans AM, Dai H, Lawton KA., Metabolon, Inc. J Cheminform. 2010 Oct 18;2(1):9.

DiseaseMechanism

DNA

RNA

Proteins

MetabolismL-threonine

NH2

OH O

O H

glucose

OH

OH

OHOH

OOH

cholesterol

MetaboLoN PLatFoRM teChNoLoGy

DRuG vs vehICLe IN 24 houR uRINe CoLLeCtIoN 595 compounds detected across all urine samples.

significant effects at all doses and increasing with dose.

strong changes at high dose dominated by decreases.

GLyCosuRIa, aMINoaCIDuRIa, uReMIa amino acids decreased in plasma and concomitantly increased in urine.

amino acids are typically retained in plasma and do not appear in urine in large amounts.

significant increase of glucose (11.7-fold) in urine.

accumulation of creatinine, urea and allantoin in plasma.

both plasma and urine data clearly indicated acute and significant renal dysfunction.

eaRLy bIoMaRKeRs IN PLasMa Classical plasma markers for renal dysfunction such as urea and creatinine do not appear to be sensitive early indicators.

better renal toxicity markers would include: appearance at lower dose, appearance at earlier time, and stronger and more easily measured change.

the most significant marker 3Is is possibly linked to the mechanism of toxicity and was further developed as a biomarker test for this type of drug related toxicity.

Most sIGNIFICaNt MaRKeR: 3-INDoxyL suLFate & P-CResoL suLFate

tRyPtoPhaN CataboLIsMvarious tryptophan metabolites were elevated in plasma. tryptophan catabolism is associated with kidney and liver toxicity and Is has been specifically been linked to inducing kidney toxicity.

CoNCeNtRatIoNs oF 3-Is after a single Dose administration of Compound a, Compound b (related chemical analog with significantly reduced toxicity) and vehicle ControlAverage plasma and brain concentrations of 3IS following administration of SCH 900424 (A) and SCH 900765 (B) in 0.4% HPMC. Plasma and brain concentrations are shown following a single dose administration to CD-1 mice, indicating 3Is concentrations were significantly elevated not only in the mouse plasma but also the brain of animals treated with 100 mg/kg sCh 900424. Flat lines indicate zero concentration.

Assay Development: G. Wang and W. Korfmacher. Rapid Communications in Mass spectrometry. 2009, 23: 2061-2069.

suMMaRy oF FINDINGs histopathologic analysis indicated that toxicity involved both the renal tubules of the cortex and/or medulla (distal tubular dilation and proximal tubular degeneration) and the renal papilla (papillary necrosis and papillary casts) in all dose groups ranging from 20 to 100 mg/kg.

Metabolon analysis of urine and plasma clearly indicate acute and significant renal toxicity and 3Is as a potential strong mechanistically linked biomarker of acute renal failure.

Time (hr)

Mean 3-Indoxyl Sulfate Brain

Conc

. (ng/g

)

0

200

400

600

800

0.4% HPMC (Vehicle) 20 mg SCH 900424/kg100 mg SCH 900424/kg100 mg SCH 900765/kg

1 4 8 24Time (hr)

Mean 3-Indoxyl Sulfate Plasma Conc. (

µg/mL)

0

10

20

30

40

0.4% HPMC (Vehicle) 20 mg SCH 900424/kg100 mg SCH 900424/kg100 mg SCH 900765/kg

1 4 8 24

Plasma Brain

abstRaCtsCh900424 (compound a), a nicotinic acid receptor (NaR) agonist showed promising potent activity and was investigated for toxicology. the resulting toxicology study revealed acute renal failure and a metabolomics study was undertaken to better mechanistically understand the aRF effect and potentially identify early biomarkers of aRF. 8 mice per group were treated at 3 doses and 4 time points and a global metabolomics study was undertaken on the blood and urine of each mouse. the resulting global metabolomics study revealed 3-indoxyl sulfate (3Is) as the most sensitive and dynamic biomarker of sCh900424-induced renal toxicity. to further validate 3Is as a biomarker of aRF, a specific targeted assay was developed for measuring blood and brain levels of 3Is in response to drug dosing and additional follow-up drug dosing studies were undertaken. In addition, based on the metabolomic analysis other significant plasma markers including p-cresol-sulfate and tryptophan catabolites (kynurynate, kynurenine, 3-indole-lactate) might be of toxicological importance but have not been studied in detail. this comprehensive approach that includes untargeted metabolomic and targeted bioanalytical sample analyses could be used to investigate toxicity of other compounds that pose preclinical or clinical development challenges in a pharmaceutical discovery and development.

stuDy DesIGN FoR DRuG-INDuCeD aCute ReNaL toxICIty Mice in groups of 8 were treated with the drug in three doses and vehicle.

Plasma samples were collected at four time points (128 samples).

urine samples were collected over 24 hours (32 samples).

otheR MetaboLoN toxICIty PaPeRs ethylene Glycol Monomethyl ether-Induced toxicity is Mediated through the Inhibition of Flavoprotein Dehydrogenase enzyme Family

Discovery of Metabolomics biomarkers for early Detection of Nephrotoxicity

effects of Mainstream Cigarette smoke on the Global Metabolome of human Lung epithelial Cells

Metabolomic Profiling Can Predict Which humans Will Develop Liver Dysfunction when Deprived of Dietary Choline