University of North Carolina Drug Induced Liver Injury: Implications in drug discovery and development Paul B. Watkins University of North Carolina Chapel Hill, N.C.

Why clinical drug development programs were terminated in 1991 % of total terminations Nature Reviews: Drug Discovery, Aug, 2004

Why clinical drug development programs were terminated in 2000 % of total terminations 1991 2000 Nature Reviews: Drug Discovery, Aug, 2004

Industry SAE Priorities 2006 Rank Order [1 highest to 5 lowest] Overall Priority Variance Your Company's Priority Hepatotoxicity 1.1 low 1.2 QT Prolongation 2.6 moderate 2.5 high Rhabdomyolosis 3.3 3.5 mod Serious Skin Rashes [SJS] 3.4 Edema 4.4 4.5 SAE Consortium Survey – courtesy of Arthur Holden

Drug Induced Liver Injury (DILI) is Hot FDA / Pharma steering committee Several Critical Path Initiatives $ millions spend in industry New Network (DILIN) SAE Consortium

Regulatory actions due to DILI (1995-2007) Withdrawals bromfenac troglitazone pemoline Second Line felbamate tolcapone trovafloxacin Warnings acetaminophen leflunomide nefazodone nevirapine pyrazinamide/rifampin terbinafine valproic acid zifirlukast atomoxetine interferon 1b –1b and 1a saquinavir infliximab bosentan telithromycin (kava, lipokinex) http://www.fda.gov/medwatch/safety.htm

“Hepatotoxicity has been the most common single adverse effect causing major drug problems, including withdrawals and refusals to approve” Bob Temple, M.D. FDA 2/15/01

New Hepatotox Initiatives Liver Toxicity Biomarker Study – BG Medicine Biomarker Consortium – Hepatoxicity group The SAE consortium

Safe pathways Drug elimination Reactive Metabolite Necrosis Serum ALT

2007 State of the Art How to avoid hepatotoxicity in drug development 1). Avoid certain molecular structures

Compound Pair Ibuprofen Ibufenac “Clean” Compound “Toxic” Compound* *withdrawn from the market in the 1960’s because of clinical liver toxicity

2007 State of the Art How to avoid hepatotoxicity in drug development 1). Avoid certain molecular structures 2). Target daily dose to < 10 mg/day

2007 State of the Art How to avoid hepatotoxicity in drug development 1). Avoid certain molecular structures 2). Target daily dose to < 10 mg/day 3). Low covalent binding in liver microsomes 4). Low production of glutathione conjugates

2007 State of the Art How to avoid hepatotoxicity in drug development 1). Avoid certain molecular structures 2). Target daily dose to < 10 mg/day 3). Low covalent binding in liver microsomes 4). Low production of glutathione conjugates 5). Wide exposure margin in preclinical species.

2007 State of the Art How to avoid hepatotoxicity in drug development 1). Avoid certain molecular structures 2). Target daily dose to < 10 mg/day 3). Low covalent binding in liver microsomes 4). Low production of glutathione conjugates 5). Wide exposure margin in preclinical species. 6). Low incidence (<5%) of ALT > 3 X ULN in clinical trials.

Acetaminophen fails every criteria 1). Avoid certain molecular structures - No 2). Target daily dose to < 10 mg/day – 4 Gm/day 3). Low covalent binding in liver microsomes - No 4). Low production of glutathione conjugates - No 5). Wide exposure margin in preclinical species – No 6). Low incidence (<5%) of ALT > 3 X ULN in clinical trials. No

JAMA, 392:87,2006

Tylenol kills pain, may hurt liver, is harmless to oxen   Written by Frank Cotolo                                                                     Bad cells in the liver attacking good cells. Dr. Paul "Bennies" Watkins … lead author of the study, said … "This Tylenol thing is nothing new. The liver is an important organ. That, also, is nothing new. The liver is a strong organ. That is nothing new. Tylenol is not strong enough to destroy a liver. That is debatable. Debates do not prove anything. That is nothing new. Debates about drugs and studies about drugs cost money because none of us researchers like to work for nothing. That is nothing new. http://www.thespoof.com/news/spoof.cfm?headline=s5i11002

Drug Induced Liver Injury (DILI) 1). Thorn in the side of drug development. 2). High priority to design out of drugs. 3). Little progress made to date.

Why is human hepatotoxicity so difficult to predict from preclinical studies?

A potential mechanism of DILI not related to reactive metabolite Bile canaliculus BSEP Bile acids Hepatocyte Hepatocyte

Drug Induced Liver Injury (DILI) can mimic every known liver disease Cholestasis (&vanishing bile duct syndrome) Steatosis (micro and macrovesicular) Phospholipidosis Veno-occlusive disease Occult fibrosis/ cirrhosis Liver cancer Acute hepatocellular injury – High ALT/AST

Regulatory actions due to DILI (1995-2007) Withdrawals bromfenac troglitazone pemoline Second Line felbamate tolcapone trovafloxacin Warnings acetaminophen leflunomide nefazodone nevirapine pyrazinamide/rifampin terbinafine valproic acid zifirlukast atomoxetine interferon 1b –1b and 1a saquinavir infliximab bosentan telithromycin (kava, lipokinex) http://www.fda.gov/medwatch/safety.htm

Of the 23 drugs/CAM that have undergone withdrawal, restriction or warnings 19/23 (82%) were associated with acute idiosyncratic hepatocellular injury

“idiosyncracy” (Hippocrates, ~400 B.C.) John Senior - FDA (idios) - one’s own, self (syn) - together (crasis) - a mixing, mixture therefore a person’s own mixture of characteristics, factors, nature and nurture, uniquely John Senior - FDA

SAFE Liver injury ( ALT) safe Concept of idiosyncratic hepatocellular injury

death jaundice enceph Days on drug 1). Orient to axis – note log scale – credit John Senior 2). Normal values until 3 months, then AST/ALT, next symptoms, then jaundice, then death 3). Fall in AST/ALT due to loss of liver not improvement – drug stopped too late. 4). Pattern more or less consistent across drugs, but signature features exist. 5). Rare to see this in a clinical trial

Challenges in identifying factors underlying susceptibility to DILI 1). How to identify susceptible individuals. 2). What to do with them once you have them.

Selection of patients based on serial ALT values in a clinical trial C.V. Non-susceptible susceptible ULN

A genetic test that predicts ALT elevations: 1). Would obviate need for ALT monitoring. 2). Would be useful in developing next in class drugs. 3). May provide only limited insight into mechanisms of idiosyncratic severe DILI.

Problem with ALT elevation as the endpoint 1). Occurs with drugs that do not have clinically important liver toxicity 2). Usually reverse with continued treatment even with drugs that can cause acute liver failure.

Incidence of ALT elevations (>3X ULN) and clinical hepatitis troglitazone 2% <0.1 INH 15% <1% diclofenac 3% <0.01%

Treatment with tacrine through ALT elevations unpublished

Reversed on treatment Treatment stopped unpublished

Reversal of rat liver necrosis with continued exposure to BDCM 1 week 3 weeks Toxicol. Sci. 64:268 (2001)

Possible explanations for reversibility of ALT elevations 1). ALT elevations that reverse on treatment have no relationship to those that can progress to liver failure. 2). A subset of those with ALT elevations can progress on to liver failure (i.e. those who can not adapt).

X Adaptation Reactive Metabolite Safe pathways Drug elimination Progressive injury X ALT elevations Adaptation

SAFE liver failure Increased ALT safe jaundice SAFE liver failure Increased ALT safe Concept of idiosyncratic hepatocellular injury

NAPQI safe elimination APAP Covalent binding/oxidative stress resolution progression

Effect of 8 days APAP pretreatment (---) on single dose toxicity in mice Hepatology 29:436, 1999.

3-Cys-APAP adducts (brown) 2 hours after single toxic APAP dose Saline pretreatment APAP pretreatment

Changes in APAP metabolism that reduce toxicity APAP elimination ROS (Nrf-2) Regeneration CYP2E1 CYP1A2 CYP2B GST GSH Acute phase (IL-6) NAPQI

Transporters during recovery from APAP hepatotoxicity TNFa X X

Transporters during recovery from APAP hepatotoxicity

Ntcp and Mrp4 expression 48 hours after APAP Alkunes and Manatou, unpublished observations

MDR1 (P-glycoprotein) expression In submassive necrosis (human) Normal liver necrosis J Pathol 200:553, 2003

submassive necrosis (human) MRP3 MRP3 expression in submassive necrosis (human) Normal liver necrosis J Pathol 200:553, 2003

Conclusion Adaptation to liver toxicity can involve: a). Down regulation of CYPs and uptake transporters b). Upregulation of glutathione and efflux transporters

Serial ALT in healthy woman receiving APAP 1 g qid X 7 days Unpublished data

NAPQI safe elimination APAP Covalent binding/oxidative stress resolution progression innate immune system

Balance Between Pro and Anti-inflammatory Cytokines Kaplowitz, 2005, Nature Review Drug Discovery

Balance Between Pro and Anti-inflammatory Cytokines Kaplowitz, 2005, Nature Review Drug Discovery

Conclusions Adaptation is probably most important issue in idiosyncrasy. a). Determines whether patient gets sick b). Implications for monitoring b). Susceptibilities may not be drug specific Current concepts do not account for the “memory”.

Where do we go from here? The most appropriate model for studying idiosyncratic hepatotoxicity are the people who actually experienced severe toxicity (i.e. they lacked the ability to adapt to the toxicity).

Selection of cases and controls from serial ALT values in a clinical trial C.V. Non-susceptible susceptible ULN

A cooperative agreement funded by the Division of Digestive Diseases and Nutrition, National Institute of Diabetes and Digestive and Kidney Diseases http://dilin.dcri.duke.edu/

Sphere of Influence 12.8 million lives http://dilin.dcri.duke.edu/

Resources Created by DILIN 1). Genomic DNA bank. 2). Immortalized lymphocyte bank. 3). Registry of subjects.

Final take home points 1). The DILIN network represents the best opportunity to date to identify mechanisms underlying severe idiosyncratic DILI. 2). Research utilizing the resulting resources will be challenging.

Idiosyncratic hepatocellular injury due to drugs is a model for all environmental disease 1). Large population with known “exposure” to a defined xenobiotic (the drug). 2). Biomarkers that are cheap, safe, and sensitive.