Transcript Blue sky: early predictability for DILI

New biomarkers for drug-induced liver injury:
First insights from clinical qualification
Joint Conference of European Human Pharmacological Societies,
Nice, April 11-12, 2013
Michael Merz, Novartis Institutes for BioMedical Research
Outline
 DILI background
 Shortcomings of current liver safety biomarkers,
requirements for advanced markers
 Biomarker qualification in the IMI SAFE-T consortium
 Initial findings for new liver safety biomarkers
 Biomarker discovery
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New biomarkers for drug-induced liver injury - M Merz - April 11, 2013
Drug safety: room for improvement
Attrition in drug development
Success rate [%]
• Around 90% of compounds entering
clinical development fail
Development phase
• 30% of these failures are due to
clinical safety and toxicology
Kola et al. (2004), Nat Rev Drug Discovery; 3: 711-15
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Attrition rate over time
A universal and eternal constant?
Nat Rev Drug Discovery 2012; 11(1): 17-18
• Do we have to live with losing
e.g. 1/3 of drugs in phase III
and 1/6 during submission??
Reasons for withdrawals
Probability of success by development phase
• For decades, drug-induced liver injury has been
one of the key safety issues in drug development
and post marketing
Drug Info J 2001; 35:293
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Withdrawals due to drug-induced liver injury (DILI)
Reducing treatment options for key disease areas
Withdrawals
1970
Ibufenac
1959
Iproniazid
1962
Thalidomide
1967
Oxyphenisatin
1982
Benoxaprofen
Ticrynafen
1985
Perhexiline
1984
Methaqualon
1991
Triazolam
1996
Alpidem
1997
Fenfluramine
Tolcapone
Tolrestat
1998
Terfenadine
Bromfenac
2007
Lumiracoxib
2006
Ximelagatran
2005
Pemoline
2004
Rofecoxib
2000
Alosetron
2003
Cisapride
Nefazodone
Amineptine
Troglitazone 2001
Cerivastatin
Trovafloxacin
Diabetes
CNS disorders
RA/OA
Cardiovascular disorders
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New biomarkers for drug-induced liver injury - M Merz - April 11, 2013
Constipation
Infections
Drug-induced liver injury (DILI)
Key challenges
 DILI is the leading cause of acute liver failure in the United States
 In the post-approval setting, DILI is a leading cause of regulatory actions, including
drug withdrawals, label changes and boxed warnings
 Across the industry, we regularly loose promising candidates due to DILI
• A part of those may be false positives
 Of predominant concern are idiosyncratic, hepatocellular types of DILI
• Non-dose dependent (?)
• Not predictable (as yet)
• High rate of liver failure, often fatal outcome
• Rare
 A major issue is the lack of suitable markers allowing for
• Early signal detection
• Mechanistic assessment
• Robust prediction of clinically relevant effects
• Risk assessment in individual patients
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New biomarkers for drug-induced liver injury - M Merz - April 11, 2013
Standard liver tests: ALT, AST, AP, gGT, bilirubin
Some shortcomings
 Inadequate sensitivity and specificity
 Limited predictive value, both from a translational and clinical outcome perspective
 Do not allow for differentiation between injury, upregulation, reduced clearance
 Half life of aminotransferases too long to allow for close monitoring and
assessment of rapid changes in liver status
 Aminotransferase activities frequently confounded by e.g. effect of different diets
and different levels of physical exercise
 Not supporting mechanistic understanding
 Focusing on liver only, not taking into account immune system involvement
Clear need for alternative biomarkers of drug related liver injury.
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New biomarkers for drug-induced liver injury - M Merz - April 11, 2013
Specific liver test attributes of interest
 Patient level
– Lower injury threshold
– Earlier time to onset
– Larger extent of changes
– Improved liver specificity
– Better suited to monitor and predict clinical outcome
– Better suited to assess causality
 Population level
– Earlier and more specific liver signal detection in clinical
development programs
– Improved mechanistic insight
– Superior in terms of identifying underlying pathology
– Better suited to predict human risk from animal toxicity
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New biomarkers for drug-induced liver injury - M Merz - April 11, 2013
Key challenges for biomarker qualification
 Substantial background variability in initial candidate markers
 Biomarker response varies across different populations
 Large initial number of biomarker candidates requires substantial
sample volumes to be taken
 Cases with key target response, i.e. DILI, suitable and accessible for
qualification, are overall very rare
 Large sample sizes are required
 Multitude of patient populations need to be included
Qualification cannot be achieved by one company alone
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New biomarkers for drug-induced liver injury - M Merz - April 11, 2013
IMI SAFE-T Consortium
Objectives
 To evaluate utility of safety biomarkers for detecting, assessing, and
monitoring drug induced kidney, liver, and vascular injury in humans
 To develop assays and devices for clinical application of safety
biomarkers
 To compile enough evidence to qualify safety biomarkers for regulatory
decision making in clinical drug development and in a translational context
 To gain evidence for how safety biomarkers may also be used in the
diagnosis of diseases and in clinical practice
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SAFE-T participants
Academia
SMEs
Advisors
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Collaborators
SAFE-T Biomarker qualification process
Elements and process flow
Literature
Q2 2009
DILI BM step 1 list
Select
Evaluation
Databases
DILI BM step 2 list
SAFE-T sources
Q1 2010
Regulatory advice
Assay availability / development
Exploratory
phase
Select
DILI BM step 3 list
Assay / stat analysis / select BMs
Healthy volunteers
Patients non-liver disease
Background
variability
DILI BM step 4 list
Assay / stat analysis / select BMs
Samples
Thresholds
Q2 2011
Patients hepatotoxic drugs
Confirmatory
phase
Patients liver disease
Regulatory advice
Assay / stat analysis / select BMs
Qualification
DILI BM f inal list
Q2 2014
Submit to health authorities
Regulatory approval
12 New biomarkers for drug-induced liver injury - M Merz - April 11, 2013
DILI biomarker selection process
Two (& a half) stage approach
Exploratory phase
Stage gate
analysis
Confirmatory phase
Drop
Background
variability
Drop
Information on...
Drop
Response to DILI
Pathology?
Response to nonliver disease
Mechanism?
Disease severity?
Response to nonDILI liver disease
Drug-relatedness?
Clinical outcome?
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Ongoing prospective clinical studies
Populations and (some) key objectives
 Multi-center international study in patients with suspected drug-induced liver injury
• Sensitivity, specificity, predictive value (outcome), association with standard markers, time profiles
 Single-center study in rheumatoid arthritis patients
• Specificity, association with standard markers
 Single-center study in patients with acute or myeloid lymphoblastic leukemia on chemotherapy
• Sensitivity, specificity, predictive value (outcome), association with standard markers, time profiles
 Multi-center study in patients after liver transplantation
• Link to histopathology, association with fibrosis progression
 Multi-center study in patients on antituberculosis treatment
• Differentiation of susceptible patients from adaptors and tolerators
 Multi-center Swiss study in patients with suspected drug-induced liver injury
• Specificity, predictive value, association with genetic suspectibility markers
 Single-center study in nevirapine-treated HIV patients
• Differentiation of susceptible patients from adaptors and tolerators
 Single-center study in acetaminophen-overdose patients
• Predictive value (outcome), association with standard markers, time profiles, mechanistic understanding
14 New biomarkers for drug-induced liver injury - M Merz - April 11, 2013
Biomarker candidates
Initial selection
Candidate markers
Liver specificity
Pathology
microRNA 122
Highly specific
Hepatocellular injury
albumin mRNA
Highly specific
Hepatocellular injury
Microglobulin precursor (Ambp) mRNA
Highly specific
Hepatocellular injury
High mobility group (HMGB-1), hypo- and hyperacetylated forms
Not specific
Hepatocellular injury, inflammation
Cytokeratin 18, full length and caspase cleaved fragment
Not specific
Hepatocellular injury, necrosis vs apoptosis
Highly specific
Cholestatic injury
Not specific
Cholestatic injury, biliary hyperplasia
Highly (ALT1)
Hepatocellular injury
Glutamate dehydrogenase (GLDH)
Specific
Hepatocellular injury
Purine nucleoside phosphorylase (PNP)
Specific
Hepatocellular injury
Not specific
Hepatocellular injury
Specific
Hepatocellular injury
F-protein (HPPD)
Highly specific
Hepatocellular regeneration
Arginase 1
Highly specific
Hepatocellular injury
alpha-fetoprotein
Specific
Hepatocellular injury
Regucalcin
Specific
Hepatocellular injury
alpha2,6-sialyltransferase (ST6gal)
Specific
Inflammation
Osteopontin
Not specific
Inflammation
Colony stimulating factor receptor (CSF1R)
Not specific
Inflammation
Specific
Hepatocellular function
Not specific
Inflammation
Conj./unconj. bile acids
Urocanic Acid
ALT1 & 2
Malate dehydrogenase (MDH)
Glutathione S-Transferase (GST-alpha)
Paraoxonase 1 (PON1)/Prothrombin
Leucocyte cell-derived chemotaxin2 (LECT2)
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Blood-based microRNA biomarkers for DILI
Evidence from preclinical models
Courtesy Ina Schuppe Koistinen, Astra Zeneca
miR-122
miR-192
ALT
miR-122
 Liver tissue specific
 Translatable to human
 Earlier detection than ALT; greater sensitivity; less variability...
APAP/mouse
mir-122
mir-192
ALT
APAP/mouse
Yi Zhang et al, Clin Chemistry, 2010
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Wang et al, PNAS, 2009
Serum microRNAs as human DILI biomarkers
Specificity of miR-122 for liver injury
Courtesy Jonathan Moggs, Novartis
ALT
miR-122
***
***
1000000
miR-192
miR-1
miR-218
***
***
***
10000
***
*
ALI: acute liver injury
100
CKD: chronic kidney disease
APAP: acetaminophen
non-APAP ALI:
- autoimmune
1
- HBV
- HCV
- Clarithromycin DILI
17 New biomarkers for drug-induced liver injury - M Merz - April 11, 2013
APAP ALI
NON-APAP ALI
CKD
APAP NO ALI
Healthy Controls
APAP ALI
NON-APAP ALI
CKD
APAP NO ALI
Healthy Controls
APAP ALI
NON-APAP ALI
CKD
APAP NO ALI
Healthy Controls
APAP ALI
CKD
NON-APAP ALI
APAP NO ALI
Healthy Controls
APAP ALI
CKD
NON-APAP ALI
Starkey-Lewis et al., (2011)
Hepatology 54:1767
APAP NO ALI
0.0001
Healthy Controls
0.01
Standard liver tests: focus on liver only
Need to account for mechanistic background
Drug
Immune system markers
Hepatocyte/cholangiocyte markers
Adapted with permission from Kaplowitz N, Nat Rev Drug Discov. 2005 Jun; 4(6): 489-99
18 New biomarkers for drug-induced liver injury - M Merz - April 11, 2013
HMGB1 and Cytokeratin 18
Mechanism based biomarkers
Slide courtesy Neil French, MRC CDSS
Necrosis and Inflammation:
• HMGB1 – chromatin binding protein
• Passive release by necrotic cells
• Active release by activated immune cells
(hyper-acetylated (Lys NLS))
• Cytokine activity (TLR/RAGE)
Apoptosis:
• Keratin-18 – intermediate
filament protein / structural
integrity
• Is cleaved by caspases
• Fragment released into blood
• Full length K18 passively released
during necrosis
Antoine DJ et al., 2010 Mol Med
Antoine DJ et al., 2009 Toxicol Sci
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Patients post acetaminophen overdose
Markers for inflammation, necrosis, and apoptosis
Based on Antoine DJ et al., 2012 J Hepat
Association with King‘s College Criteria for prognosis of acute liver failure
 Acetylated HMGB1 may be a prognostic DILI marker, indicating extent of inflammation
 Caspase cleaved cytokeratin 18 may have value as a prognostic DILI marker, indicating
involvement of apoptosis as protective mechanism
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Diagnostic value of Glutathione S transferase a (aGST)
 Glutathione S transferase a (aGST):
• Inducible phase II detoxification enzyme
• Four isozymes of GST expressed in human and other mammals; aGST is a liver specific
dimer expressed in human hepatocyte cytosol
• High concentration in centrilobular cells: may be more sensitive than ALT and AST
• Half life ~1 h in humans: may be useful for close monitoring
• Low molecular weight: release into plasma may occur earlier than for ALT and AST
 Meta-analysis of four Novartis phase 1 studies using aGST for liver monitoring
• 150 healthy subjects (108 m, 42 f), age 18 - 60, BMI 18 -32, duration 1-4 weeks
• Key objectives:
o Analyse correlation of aGST levels with age, BMI, and aminotransferases at baseline, and with
aminotransferases during treatment (active drug and placebo)
o Characterize time profiles of aGST as compared to ALT and AST
o Explore to which extent aGST levels may be able to support causality assessment in case of elevated
aminotransferases.
21 New biomarkers for drug-induced liver injury - M Merz - April 11, 2013
Diagnostic value of Glutathione S transferase a (aGST)
Preliminary results from Novartis meta-analysis
• Earlier onset and faster resolution?
• Helpful for causality assessment in a subset of cases?
Treatment end
ALTn
GSTn
ASTn
• Time to onset of enzyme elevations may be marginally shorter with aGST
• In some patients, aGST returns to baseline faster , possibly supporting causality assessment
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Initial, preliminary results of stage gate samples
CK18 full length and fragment, MCSF-R: DILI association
*
*MCSF-R=CSF1R:
cytokine receptor, controlling
macrophage proliferation and function
 Significant associations with DILI
23 New biomarkers for drug-induced liver injury - M Merz - April 11, 2013
Initial, preliminary results of stage gate samples
CK18 full length and fragment, MCSF-R: DILI (and gender?) association
 MCSF-R: association with DILI and gender?
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Initial, preliminary results of stage gate samples
CK18 full length and fragement, MCSF-R: absence of age dependency
 No associations with age
25 New biomarkers for drug-induced liver injury - M Merz - April 11, 2013
Initial, preliminary results of stage gate samples
CK18 full length and fragement, MCSF-R: absence of BMI dependency
 No associations with BMI
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Parallel to qualification: DILI biomarker discovery
Why?
 Biomarker candidates do not cover all objectives of SAFE-T DILI WP
• Lack of susceptibility markers
• Lack of sensitive functional markers, some pathologies poorly represented
• Most markers identified in pre-clinical models
How?
 Based on human DILI cases from SAFE-T clinical studies
 Characteristic changes in serum proteome and metabolome expected
• Mass spec and protein antibody array analyses of plasma samples ongoing
27 New biomarkers for drug-induced liver injury - M Merz - April 11, 2013
Watkins Study: early predictive DILI markers
Study design and initial output
Courtesy Ina Schuppe Koistinen, AstraZeneca
Healthy men and women (18-55 years) were treated with 4g acetaminophen/day for 7 days
17 subjects:
responders (ALT >2.0 x baseline level)
15 subjects :
intermediate responders (ALT 1.5-2.0 x baseline level)
18 subjects:
non-responders (ALT <1.5 x baseline level)
Results
 Urine metabolite profiles prior or at start of
treatment not predictive of DILI
 Urine profiles at day 5-6 (prior to raised ALT)
could distinguish responders from nonresponders
 Predictive metabolites include APAP and
endogenous metabolites
Winnike JH, Li Z, Wright FA, Macdonald JM, O'Connell TM, Watkins PB. Use of
pharmaco-metabonomics for early prediction of acetaminophen-induced hepatotoxicity in
humans. Clin Pharmacol Ther. 2010;88(1):45-51
O'Connell TM, Watkins PB. The application of metabonomics to predict drug-induced liver
injury. Translational Medicine, 2010, 88(3): 394-99
28 New biomarkers for drug-induced liver injury - M Merz - April 11, 2013
Watkins Study: early predictive DILI markers
Additional benefit of serum proteomics and metabolomics
Courtesy Ina Schuppe Koistinen, AstraZeneca
• LC-MS based profiling for compound and endogenous metabolites
• Suspension bead protein array (antibody based)
• Endogenous metabolites and
Metabolomics
protein profiles identified, that:
• Predict ALT elevations at baseline
Proteomics
PLS-DA 2
treatment
PLS-DA 2
(susceptibility markers)
• Predict ALT elevations early during
• Pathways involved
time of ALT elevations
PLS-DA 2
PLS-DA 2
• Cell death pathways activated at the
PLS-DA 1
PLS-DA 1
• Pyruvate and glutamate metabolism at
baseline
Day 5 and 6
Pre-dose
• Overlap with ximelagatran candidate
biomarkers
PLS-DA 1
Day 8 and 9
29 New biomarkers for drug-induced liver injury - M Merz - April 11, 2013
PLS-DA 1
Day 12
Conclusions
 Advanced safety biomarkers for prediction, detection, and assessment of druginduced liver injury (DILI) are urgently needed.
 Due to the low incidence of DILI, large sample sizes across a range of different
populations are required for clinical qualification of new markers.
 Large scale public private partnerships involving industry, academia, small to
medium sized enterprises, and regulators such as the IMI SAFE-T consortium
may be the most efficient way to successful biomarker qualification.
 A list of promising DILI biomarker candidates has been selected by the SAFE-T
consortium for clinical qualification.
 Preliminary data on a subset of markers offer first insights into potential
predictive and diagnostic value.
 Regulatory approval of new DILI biomarkers with defined contexts of use is
expected by 2015.
30 New biomarkers for drug-induced liver injury - M Merz - April 11, 2013
Acknowledgements
Neus Prats
Marc Walton
Axel Kretschm er
Denise Robinson-Gravatt
Martin Shaw
Elisabetta Cargnello
Anna Dom ènech
Hüseyin Firat
Thom as Krahn
Bernard Souberbielle
Corm ac Kilty
Nicole Schneiderhan-Marra Ahm ed Sheriff
Ina Naum ov
Eric Massana
Kaïdre Bendjam a
Nicolas von Behr
David Reynolds
Vicky McGrath
Hugo Häm m erle
Rolf Seeland
Ronni Gam zu
Pere Berga Marti
Peter Thom ann
Heidrun Ellinger-Ziegelbauer Robin Privm an
Barry Hayes
Günter Beck
Theresa Klein
Bernd Stow asser
Mariona Auli
Attila Garam i
Matthias Gottw ald
Dom inique Brees
Rory Connolly
Thom as Schw eikert
Thom as Berg
Isabelle Clavier
Silvia Lopez
Béatrice Molac
Susanne Schw enke
Ceri Collins
Mark Pinches
Jens Göpfert
Florian van Böm m el
Frederic Galli
Jose-Luis Diaz
Fuat Firat
Katja Matheis
Erik Kuja
David Brott
Stefanie Rim m ele
Stefanie Frank
Joachim Tillner
Jam es Matcham
Stella Suzanne
Christine Rentzsch
Andrew Zehner
Ina Schuppe Koistinen Hannes Planatscher
Sabine Boas-Knoop
Xavier Benain
Alexandre Mencik
Brice Suire
Uw e Bam berger
Shelli Schom aker
Håkan Andersson
Frank Dieterle
Lina Badim on
Joost Dw erhagen
Sim on Gibbs
Barbara Fischer
Birgit Stierstorfer
Cornelia Ciorciaro
Anders Sam uelsson
Peter Hoffm ann
Teresa Padro
Isabelle Pirani
Patrice Cacoub
Dorina Bratfalean
Arno Kalkuhl
Laura Suter Dick
Sally Price
Michael Merz
Xavier Sánchez-Vallve
Catherine Lunven
David Saadoun
John Haselden
Joachim Stangier
Piotr Szczesny
Jesper Hedberg
Robert Schm ouder
Nuria Nadal
Olivier Pasquier
Dam ien Sene
Andrew Nicholls
Kristiane Wetzel
Barbara Lenz
Björn Glingham m ar
Ursula Knauf
Gem m a Vilahur
Jean-Charles Gautier
Michel Buchert
Elaine A. Irving
Dr. Volker Mahlbacher
Jacky Vonderscher
Pierre Dönnes
John Prince
Silvia Góm ez
Nathalie Piton
Anne Skrobot
Vincent Mooser
Dorothee Schw all-Rudoph
Lucette Doessegger
Terry Reed
Jeffrey Donohue
Sandrine Schw artz
Marie-Hélène Pascual
Florence Ghrenassia
Fiona J McClure
Marcus Kostka
Manfred Argast
Ann-Cathrine Jonsson-Rylander
Francois Legay
Daniel Sanchez
Muriel Breitbach
Thierry Poynard
Aubrey Sw ain
Nadine Erne
Christoph Wandel
Li-Ming Gan
David Laurie
Thom as Joos
Sylvie Brohier
Mona Munteanu
Theo Dare
Andreas Port
Rodolfo Gasser
Jenny McKay
Alexandre Avram eas
Martina Kahnert
Jean-François Gallas
Hugo Perazzo
Ian Rom an
Ulf Neum ann
Bernhard Risse
Jonas Bergström
Marie Anne Valentin
Jessica Mittelstädt
Reinhold Stockbrugger
Servane de Penquer
Federica Crivellente
Maxim ilian Schm eding
Rosem arie Kientsch-Engel Marcus Gry
Philip Bentley
Manfred Schm olz
Bernard Levin
Laurent Becquem ont
David How e
Eveline Fräßdorf
Thom as Schindler
Harry Southw orth
Gerd Kullak-Ublick
Thom as Knorpp
Ali Yağız ÜRESİN
Benoit Labarthe
Landry Cochard
Janine Heyder
Isabelle Gilet
Scott Adler
Franck Meyer
Derek Leishm an
Hugh Laverty
Volker Schm itz
Nadir Arber
Sif Orm arsdottir
Steve Hall
Sarah Chatham
Kevin Park
Anne Gysem bergh-Houal Marc Loher
Ralf Schindler
Ofer Eyal
Joe Keenan
Christiane Andriam androso Eckart Schott
Sara Kraus
Stephen Furlong
Stefan Sultana
Sim on Harper
Munir Pirm oham ed
Cam illa Stephens
Maribel Lucena
Patrick Murray
Colin Green
Magnus Nord
Michael Law ton
Paul Com m ander
Neil French
Brooke Bergeron
Jean-Marc Vidal
Marisa Papaluca
Raúl Andrade
Kevin Park
Neil French
Hannes Planatscher
Daniel Antoine
Jens Goepfert
Nicole Schneiderhan-Marra
Björn Glinghammar
Ina Schuppe Koistinen
Teresa Padro
Lina Badimon
Thomas Joos
Gerd Kullak-Ublick
Thierry Poynard
31 New biomarkers for drug-induced liver injury - M Merz - April 11, 2013
Jonathan Moggs