Agents that inhibit the BSEP and mitochondrial function what do we know? Prepared by Michael D. Aleo, Ph.D. Groton, Investigative Toxicology

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1 Agents that inhibit the BSEP and mitochondrial function what do we know? Prepared by Michael D. Aleo, Ph.D. Groton, Investigative Toxicology

2 Declarations Nonclinical studies All procedures performed on animals in this presentation were in accordance with regulations and established guidelines and were reviewed and approved by Pfizer Institutional Animal Care and Use Committee or its equivalent Clinical Studies were conducted in accordance with the International Conference on Harmonization guideline for Good Clinical Practice and the principles of the Declaration of Helsinki and was approved by the local independent ethics committee. All subjects gave written informed consent prior to participation in the study. SLIDE 2

3 CP (Nonclinical and Clinical Findings) Summary of Repeat-Dose IND Enabling Toxicology Studies Study Type Species Dose Levels (mg/kg/day) Cmax, total (µm) 1-Month Oral Gavage Rat (Sprague-Dawley) Month Oral Gavage Dog (Beagle) Major Liver Findings No liver clin path related or histological effects (bile acids were not measured) nonseverely toxic dose ALT, AST, ALP, GGT, total bilirubin, bile acids, cholestasis (bile plugs in centrilobular area in one dog) Summary of Human Clinical Trial Experience# From Munster et al., Clin Cancer Res 13: , 2007 SLIDE 3

4 CP Post-hoc examination of possible mechanisms Direct Cellular Risk Factors Human Hepatocytes Cytotoxic Risk Mitochondrial Risk BSEP Risk Reactive Molecule 13 µm HepG2 Glu/Gal POS Vesicles µm DansylGSH NEG Isolated Mitochondria 14 µm Cellular CLF 1.7 µm Cytotoxic P-450 Generated Metabolite Overall features: Clinical pathology and histological effects observed in dogs and not rats at higher Cmax values compare to sensitive humans (small trials) Clinical Cmax to effect ranged between µm in isolated patients Exposure near values where combined BSEP and mitochondrial inhibition occurs with direct (or metabolite driven) cytotoxicity in vitro POS SLIDE 4

5 PF (Nonclinical findings in rat and dog IND enabling studies) Summary of Repeat-Dose IND Enabling Toxicology Studies Study Type Species Dose Levels (mg/kg/day) Cmax (µm) Major Liver Findings 1-Month Oral Gavage Rat (Sprague- Dawley) µm Reversible increases in liver weights at 150 and 200 mg/kg/day correlated with microscopic hepatocyte hypertrophy Hepatic (lipid) vacuolation at > 50 mg/kg/day. 1-Month Oral Gavage Dog (Beagle) µm Reversible histopathological changes in livers at 75 and 100 mg/kg/day Minimal individual hepatocyte necrosis, minimal hepatic basophilia, mild hepatocyte atrophy, minimal to mild inflammatory cell infiltration and hepatocyte vacuolar degeneration. These changes were not associated with alterations in clinical chemistry parameters. A few dogs had large variations in ALT, ALP, TBili and triglycerides without histologic correlate. Findings may have been related to poor condition of dogs. SLIDE 5

6 PF Human experience Mean Changes in Selected Chemistry Laboratory Parameters from Baseline to Visit 6 (up to Day 29 or End of Treatment) Parameter Statistic Placebo N = 2 ALT (U/L) T 100 mg BID N = 9 T 200 mg BID N = 8 Baseline mean Visit 6 (Day 29)/End Of Treatment mean AST (U/L) Baseline mean Visit 6 (Day 29)/End Of Treatment mean GGT (U/L) Baseline mean Visit 6 (Day 29)/End Of Treatment mean Alkaline phosphatase (U/L) Baseline mean Visit 6 (Day 29)/End Of Treatment mean 57.5 (14.85) Asymptomatic elevations in transaminases occurring at 100 mg BID with evidence of cholestasis (AP and GGT elevations) occurring at 200 mg BID SLIDE 6

7 PF Post-hoc examination of possible mechanisms Direct Cellular Risk Factors Cytotoxic Risk Mitochondrial Risk BSEP Risk Reactive Molecule Human Hepatocytes 17 µm Glu/Gal Ratio NO Vesicles 50 µm DansylGSH POS Overall features: Isolated Mitochondria 11 µm Cellular CLF Isolated, non-concordant liver effects in dogs Clinical Cmax to effect ranged between µm in isolated subjects Exposure at fraction of values where combined BSEP and mitochondrial inhibition occurs with direct cytotoxicity and other potential reactive mechanisms occur in vitro Evidence of bile acid elevations (serum and urine) 5 µm changes in fractionated and total, within normal limits Cytotoxic P-450 Generated Metabolite NEG SLIDE 7

8 PF (Nonclinical and Clinical Findings) Summary of Repeat-Dose IND Enabling Toxicology Studies Study Type Species 1-Month Oral Gavage Rat (Sprague-Dawley) µm Dose Levels (mg/kg/day) Cmax, total (µm) Major Liver Findings Mean liver weight in females at all doses without treatment-related microscopic findings 1-Month Oral Gavage Monkey (Cynomolgus) µm None with regard to liver Summary of Human Clinical Trial Experience High incidence of transaminase elevations in a cohort of 8 healthy male subjects treated with 300mg B.I.D. for 2 consecutive weeks was observed, preventing further escalation of doses and even dose re-challenge. Six cases with abnormal (> grade 1) ALT values in 8 exposed subjects One case with unexpected severity with peak ALT>5 times ULN One case of abnormal ALT associated to clinically significant increases of pancreatic lipase and amylase leading to early discontinuation SLIDE 8

9 PF Post-hoc examination of possible mechanisms Risk Factors Cytotoxic Risk Mitochondrial Risk BSEP Risk Reactive Molecule Human Hepatocytes 177 µm Glu/Gal Ratio NO Vesicles 103 µm DansylGSH Equivocal Isolated Mitochondria 10 µm Cellular CLF >125 µm Cytotoxic P-450 Generated Metabolite Overall features: No liver effects in animals Clinical Cmax to effect was approximately 25 µm in subjects Exposure at fraction of values where weak BSEP inhibition (combined with more potent mitochondrial inhibition) and cytotoxicity occurs in vitro Elevated fractionated and total bile acid profile (within normal limits) Aligned with transaminase elevations in affected subjects POS SLIDE 9

10 Distribution of Dual Risk Factors and Drug Label Dual potent inhibitors stratify across FDA label for liver injury with majority in Withdrawn or BBW category in this dataset (N=70). Aleo et al., Hepatology 2014;60: SLIDE 10

11 Influence of Liver ATP levels on Human Biliary Function 60% 60% 30% 30% Bile samples were obtained from humans relieved of obstructive jaundice by percutaneous transhepatic biliary drainage and samples of liver were obtained during surgery and were measured for liver ATP content A 30% reduction in liver ATP levels is associated with a 60% reduction in biliary function (Indocyanine Green (ICG) and bile acid transport) in humans. BSEP and MRP2 are both ATP dependent transporters Chijiiwa et al., World J Surg 2002;26: SLIDE 11

12 SUMMARY Retrospective analysis shows two common themes Nonclinical IND enabling studies showed little to no evidence of liver injury occurred at high exposure values (intrinsic and relative to clinical exposure) with or without evidence of cholestasis (microscopic or clinical chemistries) Animals appeared less sensitive than humans for transaminase elevations These small molecules showed combined BSEP and mitochondrial inhibition, with or without cytotoxic potential in human hepatocytes Association vs Causation When we had access to clinical samples we showed some value in analysis of fractionated bile acids in serum. Perturbations in bile acid fractionation profile occurred with or without elevations in transaminases, without elevations in total bile acids. We know very little about variations in fractionated bile acids in the context of a clinical setting across patients and time? Do perturbations in fractionated bile acids adequately reflect isolated or combined BSEP and mitochondria inhibition occurring clinically? Need for developing a balanced perspective How many times are these features observed in other marketed products that do not exhibit human liver injury? Requires broader examination across marketed drugs to understand how to best interpret and possibly predict effects in humans Human experience is still very much needed as there are a lot of assumptions that go into predictions Variables of time, low incidence translation, and human susceptibilities (environment and genetic) need to play out SLIDE 12

13 Acknowledgments Scott Obach dansylgsh data Payal Rana P-450 cytotoxic metabolite data Cyprotex Human Hepatocytes HCI for cytotox & cellular CLF Paul Bonin/Solvo - BSEP vesicle Rachel Swiss Isolated mitochondria Laurie Warren Glu/Gal assay Jennifer Colangelo and Lina Luo fractionated bile acids Study directors, technicians and staff associated with nonclinical studies Clinicians, nurses and personnel involved in the clinical studies SLIDE 13