Circulating Liver mRNA as a DILI Biomarker - AASLD

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Circulating Liver mRNA as a DILI Biomarker - AASLD

Circulating RNA as a Biomarker• Blood mRNA used in trauma patients to predict mortalityand detect complications (not clear the form of the RNA)(Clin Chem, 2004)debrismicroRNAsHomeostasis...Injury...Necrosis...microvesicles• Fetal corticotrophin-releasing hormone mRNA inmaternal plasma associated with factor VII activity in preeclampsia.(J Thromb Haemost, 2008)• Blood miRNA profile altered following acetaminophenoverdose (PNAS, 2009)• Circulating mRNAs and miRNAs used for diagnosis andpredicting prognosis in multiple cancers:• Ovarian cancer (Gynecol Oncol, 2008)• Prostate cancer (PNAS, 2008)• Lung cancer (Clin Lung Cancer, 2009)• Melanoma (Clin Cancer Res, 1999)• Colorectal (Lab Invest, 2001)• And many more...The Hamner Institutes for Health Sciences | DILI Conference | March 25, 20102


Rat Models of Liver and Muscle InjuryStudy #1: D-(+)-Galactosamine Hepatotoxicity• D-(+)-Galactosamine ip exposure in Sprague Dawley rats• Single dose plus control (1000 mg/kg) at 24 h• n= 15 animals per doseStudy #2: Acetaminophen Hepatotoxicity• Acetaminophen gavage exposure in fasted Sprague Dawley rats• 3 Doses plus control (100, 700, and 1400 mg/kg)• 3 Time points 6, 24, 48 hr• n= 8 animals per dose per time pointStudy #3: Bupivacaine-induced Skeletal Muscle Toxicity• Bupivacaine HCL (BPVC) i.m. exposure in Sprague Dawley rats• Single dose plus control (0.5 ml, 0.5% in sterile saline) at 24 hr• n=10 animals (control); 20 animals (treated)The Hamner Institutes for Health Sciences | DILI Conference | March 25, 20106


Galactosamine Histopathological Examination• Centrilobular and periportal necrosis.• Degree of necrosis was moderate when qualitatively scored.Hepatocellular Necrosis Severity Score*Treatment 0 1 2 3 4 5Control 15Galactosamine 15*Number of animals scored.The Hamner Institutes for Health Sciences | DILI Conference | March 25, 2010


Galactosamine Induced Hepatotoxicity Concomitant with aIncrease in Serum ALT/AST and Liver-specific mRNA LevelsU/L1000010001001010 mg/kg1000 mg/kg***ALTAST***Copies / mL Plasma10000001000001000010001001010 mg/kg1000 mg/kg ********5' Alb mid Alb 3' AlbCopies / mL Plasma1000001000010001001010 mg/kg1000 mg/kg******5' Fgb mid Fgb 3' FgbCopies / mL Plasma10000001000001000010001001010 mg/kg1000 mg/kg ******5' Hp 3' Hp** p


Rat Models of Drug-Induced Liver and MuscleInjuryStudy #1: Galactosamine Hepatotoxicity• D-(+)-Galactosamine ip exposure in Sprague Dawley rats• Single dose plus control (1000 mg/kg) at 24 h• n= 15 animals per doseStudy #2: Acetaminophen Hepatotoxicity• Acetaminophen gavage exposure in fasted Sprague Dawley rats• 3 Doses plus control (100, 700, and 1400 mg/kg)• 3 Time points 6, 24, 48 hr• n= 8 animals per dose per time pointStudy #3: Bupivacaine-induced Skeletal Muscle Toxicity• Bupivacaine HCL (BPVC) i.m. exposure in Sprague Dawley rats• Single dose plus control (0.5 ml, 0.5% in sterile saline) at 24 hr• n=10 animals (control); 20 animals (treated)The Hamner Institutes for Health Sciences | DILI Conference | March 25, 201010


Acetaminophen HistopathologicalExamination• Centrilobular necrosis.• Degree of necrosis was moderate to severe when qualitativelyscored.Hepatocellular Necrosis Severity Score*Time Point Treatment 0 1 2 3 4 56 Control 8100 mg/kg 8700 mg/kg 81400 mg/kg 824 Control 8100 mg/kg 8700 mg/kg 6 1 11400 mg/kg 3 548 Control 8100 mg/kg 8700 mg/kg 71400 mg/kg 3 4*Number of animals scored.The Hamner Institutes for Health Sciences | DILI Conference | March 25, 2010


Acetaminophen Induced a Dose-DependentIncrease in Serum ALT/AST and Liver-specific mRNA Levels24 hr 48 hr100000100000 mg/kg - 24 hr100 mg/kg - 24 hr*700 mg/kg - 24 hr1400 mg/kg - 24 hr*1000010000 mg/kg - 48 hr100 mg/kg - 48 hr**700 mg/kg - 48 hr1400 mg/kg - 48 hr**U/L1000100U/L10010101ALTAST1ALTASTCopies / mL Plasma1000001000010001001010.10 mg/kg - 24hr100 mg/kg - 24hr* * *Alb Fgb Hp700 mg/kg - 24hr1400 mg/kg - 24hr* * * **Copies / mL Plasma1000010001001010 mg/kg - 48hr100 mg/kg - 48hr**Alb Fgb Hp**700 mg/kg - 48hr1400 mg/kg - 48hr** **p


Increase in Plasma Liver mRNAs Occurred DespiteDecreased Transcription in the LiverLog2 Fold Change10-1-2APAP Treatment*ControlAlbFgbHp-3***p


Rat Models of Drug-Induced Liver and MuscleInjuryStudy #1: Galactosamine Hepatotoxicity• D-(+)-Galactosamine ip exposure in Sprague Dawley rats• Single dose plus control (1000 mg/kg) at 24 h• n= 15 animals per doseStudy #2: Acetaminophen Hepatotoxicity• Acetaminophen gavage exposure in fasted Sprague Dawley rats• 3 Doses plus control (100, 700, and 1400 mg/kg)• 3 Time points 6, 24, 48 hr• n= 8 animals per dose per time pointStudy #3: Bupivacaine-induced Skeletal Muscle Toxicity• Bupivacaine HCL (BPVC) i.m. exposure in Sprague Dawley rats• Single dose plus control (0.5 ml, 0.5% in sterile saline) at 24 hr• n=10 animals (control); 20 animals (treated)The Hamner Institutes for Health Sciences | DILI Conference | March 25, 201014


Bupivacaine-Induced Myotoxicity Elicited an Increasein Serum ALT/AST but not Liver-specific mRNA LevelsU/L1000 ControlBPVC100101ALT****AST****Copies / mL Plasma10000 Control1000100101BPVCAlb Fgb Hp****p


Characterization of Plasma mRNA UsingSucrose GradientsTaqman qRT-PCRIsolate Plasmafrom Treatedand ControlAnimalsPerform InitialCentrifugation toIsolate Microvesicles(14,000x g, 45 min,repeat 2X)Perform SucroseGradient Centrifugationto SeparateMicrovesicles BySize/Density (210,000x g,19 hrs)Electron MicroscopyThe Hamner Institutes for Health Sciences | DILI Conference | March 25, 201016


Circulating mRNAs Possess Varied DensityDistribution with Galactosamine TreatmentCopies / mL Fraction100000010000010000100010010AlbControlDGALCopies / mL Fraction10000100010010FgbControlDGAL11Density of Fraction (g/mL)Density of Fraction (g/mL)The Hamner Institutes for Health Sciences | DILI Conference | March 25, 201017


Circulating mRNAs Possess Varied DensityDistribution with Galactosamine TreatmentCopies / mL FractionHp100000 ControlDGAL100001000100101Copies / mL FractionActb100000 ControlDGAL100001000100101Density of Fraction (g/mL)Density of Fraction (g/mL)The Hamner Institutes for Health Sciences | DILI Conference | March 25, 201018


Electron Micrographs of Sucrose FractionsShows Intact Microvesicles in Early FractionsFraction 1 (1.07 g/mL)ControlD-GalactosamineMicrovesiclesMicrovesicle Size Range: 50-200 nm (Control and DGal). (Bars = 0.5 mM)The Hamner Institutes for Health Sciences | DILI Conference | March 25, 201019


Electron Micrographs of Sucrose FractionsShows Cellular Debris in Middle FractionsFraction 6 (1.18 g/mL)ControlD-GalactosamineMicrovesiclesCell Debris andMisshapen VesiclesMicrovesicle Size Range: 50-200 nm (Control and DGal). (Bars = 0.5 mM)The Hamner Institutes for Health Sciences | DILI Conference | March 25, 201020


Electron Micrographs of Sucrose FractionsShows Intact Microvesicles in Late FractionsFraction 11 (1.26 g/mL)ControlD-GalactosamineMicrovesicleMicrovesicle Size Range: 50-150 nm (Control and DGal). (Bars = 0.5 mM)The Hamner Institutes for Health Sciences | DILI Conference | March 25, 201021


Size Distribution of Plasma Microvesicles isAltered Following Liver InjuryLarge shift from exosomes (100 nm(shedding vesicles?)Mean Plasma MV Diameter (nm)3002001000*ControlDGAL1.07 1.11 1.13 1.16 1.23 1.25Density of Fraction (g/mL)**** p < 0.05The Hamner Institutes for Health Sciences | DILI Conference | March 25, 201022


Conceptual Model for Heptatoxicity-InducedPlasma mRNAHomeostasisHepatotoxicitymicrovesiclesmicrovesiclesdebris10000001000000100000Control100000ControlDgalCopies RNA / mL Fraction10000100010010Copies RNA / mL Fraction1000010001001011.07 1.09 1.1 1.11 1.13 1.16 1.18 1.21 1.23 1.24 1.25Density (g/mL)11.07 1.09 1.1 1.11 1.13 1.16 1.18 1.21 1.23 1.24 1.25Density (g/mL)The Hamner Institutes for Health Sciences | DILI Conference | March 25, 201023


Analysis of Plasma mRNA Using WholeGenome Microarray AnalysisIsolate Plasmafrom Treatedand ControlAnimalsPerform Centrifugationto Isolate Microvesicles(14,000x g, 45 min,repeat 2X)Isolate RNA and AmplifyAffymetrix MicroarrayAnalysisThe Hamner Institutes for Health Sciences | DILI Conference | March 25, 201024


Whole Genome Microarray Analysis ShowsTreatment-Specific Profiles672 132 1242DGal*Based on 2-fold change and FDR < 0.05 criteria.APAP13 genes increased in both24 genes decreased in both95 genes differentially alteredThe Hamner Institutes for Health Sciences | DILI Conference | March 25, 201025


Preliminary Human Study FindingsAssessments of Circulating mRNAs in Patients Suffering from DILI(a collaborative effort with Dr. Paul Watkins)Study Design• Enrollees admitted to UNC Hospitals for suspectedDILI due to elevations in liver function tests• Plasma and serum collected daily during studyenrollment• Controls: 5 healthy male donors (blood seriallycollected daily over a 3-day period)Preliminary Analyses:Serum: Clinical chemistry analysesPlasma: quantitation of liver-specific Alb, Fgb,and Hp by qRT-PCRThe Hamner Institutes for Health Sciences | DILI Conference | March 25, 201026


Serum ALT/AST Profiles in Patients Enrolled inDrug-induced Liver Injury Study10000Day 1Day 2Day 310000Day 1Day 2Day 3ALT (IU/L)100010010AST (IU/L)1000100101Cont 1 2 3 4 5Patient ID1Cont 1 2 3 4 5Patient ID• Data presented for 5 patients: 4 males and 1 female (#4)• Enrollment period: 2-3 days• ALT/AST values displayed for enrollment period only• Patients typically enrolled >> 72 hr after highest ALT/AST elevation• Patients 1, 2, 5 – acetaminophen confirmed/likely causative agentThe Hamner Institutes for Health Sciences | DILI Conference | March 25, 201027


Circulating mRNA Levels are Elevated in PatientsAdmitted for Drug-induced Liver InjuryCopies / mL Plasma100010010Day 1Day 2Day 3Alb• mRNA values track with ALT/ASTelevations• Fold increases over controls arecomparable or greater than (Hp) ALT/ASTfold increases1Cont 1 2 3 4 5Patient IDCopies / mL Plasma10000100010010Day 1Day 2Day 3FgbCopies / mL Plasma1000100101Day 1Day 2Day 3Hp1Cont 1 2 3 4 5Patient ID0.1Cont 1 2 3 4 5Patient IDThe Hamner Institutes for Health Sciences | DILI Conference | March 25, 201028


Summary• Liver-specific mRNAs can be detected in plasma following hepatic injury.• Liver-specific mRNAs are sensitive biomarkers of hepatotoxicity that offergreater specificity than traditional serum ALT/AST measurement.• Generation of treatment-specific transcriptomic profiles in initial microarraystudies suggests that diagnosis of DILI and/or identification of the offendingdrug may be possible.• Plasma microparticle (MP) characterization demonstrated differentialpackaging of mRNAs along with alterations in MP size (shift to >100 nm)following liver injury. These findings suggests that, while non-specificdebris-associated mRNA release is occurring via necrosis, an active MPrelease not associated with necrotic cell death is also evident.• Preliminary clinical study findings indicate that liver-specific mRNAs arereproducibly increased following DILI in human patients.The Hamner Institutes for Health Sciences | DILI Conference | March 25, 201029


Work Published in HepatologyPMID: 20235334The Hamner Institutes for Health Sciences | DILI Conference | March 25, 201030


AcknowledgementsThomas Lab• Research InvestigatorBarbara Wetmore• Research AssociatesLinda PlutaReetu SinghHamner StaffEarl TewksburyOtis LyghtHamner IDSS CollaboratorsPaul WatkinsLorraine MehltretterExternal CollaboratorsDominique Brees (Pfizer)Michael Lawton (Pfizer)Joe Paulauskis (Pfizer)Jack Reynolds (JRC)FundingHamner Pilot Projects InitiativePfizerAmerican Chemistry CouncilNational Center for ResearchResources (Award # 50KR1095)The Hamner Institutes for Health Sciences | DILI Conference | March 25, 2010 31

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