Predictive Hepatotoxicity: Correlations in a Structural Database

1. Predictive Hepatotoxicity: Correlations in a Structural Database Jim Kelly Amphioxus Cell Technologies, Inc. Houston, TX Novel Methods for Detecting Hepatotoxicity June 8, 2004

2. ACTIVTox® • Unified human liver system for predictive toxicology • Patented liver cell line - C3A • Proprietary culture medium and methodology • Rigorous QA/QC • Cell based fluorescence and absorbance assays • Applicable across the spectrum of drug discovery, early to late

3. Objective - Human Liver-based High Throughput Predictive Toxicology • Ability to characterize a set of molecules with respect to human liver interactions • Choose among potential scaffolds • Rank compounds for liver liabilities • Feed back to chemistry in real time Structure-based drug design for toxicity and metabolism

4. ACTIVTox Displays the Functionality of Primary Hepatocytes • Series of Benchmarks demonstrate functionality • MEIC compounds • Liver specific toxicities • Drugs • Hormones • Enzyme Inductions • Cyps • Gluconeogenesis - TAT, PEPCK, 11bHSD

5. Inhibition of cyp3A Reduces Aflatoxin Toxicity

6. Standard Toxins

7. Simple, Multiparameter in vitro Toxicity Tests • Enzyme release (LDH, IDH) • Inhibition of Proliferation • ATP Content • Activation of Apoptosis • P-glycoprotein Inhibition • Induction, inhibition of cyp1A and cyp3A

8. Thiazolidinediones • TZDs comprise a new class of antidiabetic agents • Troglitazone, first to market was withdrawn due to liver toxicity • Pioglitazone and rosiglitazone do not seem to have this liability • ACTIVTox® identifies troglitazone as problematic

9. Troglitazone Rosiglitazone Pioglitazone Ciglitazone ACTIVTox discriminates among closely related TZDs

10. Troglitazone scores positively in the LDH assay Only troglitazone shows positive toxicity at 100µM

11. Troglitazone (100µM) is positive in every assay, including 3A induction

12. Trovafloxacin Temafloxacin Enoxacin Sparfloxacin Ciprofloxacin Grepafloxacin ACTIVTox distinguishes among fluoroquinolone antibiotics

13. ACTIVTox identifies Trovafloxacin

14. Trovafloxacin is Toxic at Therapeutic Concentrations

15. ACTIVTox displays fine structural discrimination among NSAIDS • Celecoxib, rofecoxib, sulindac - structurally related compounds with very different actions. Celecoxib Rofecoxib Sulindac

16. Inhibition of cyps Alters Toxicity of Celecoxib Celecoxib is a substrate for 2C9, 2D6, 3A4

17. Rofecoxib Is Not Metabolized

18. Rofecoxib does not activate Apoptosis

19. Only sulindac induces cyp1A

20. Three Compounds have Different Effects • Rofecoxib is not metabolized and does not affect either viability or expression. • Celecoxib is heavily metabolized. • Parent compound is most toxic • Activates apoptosis via inhibition of Akt signaling; central signaling pathway • Sulindac is converted to the sulfide. • Induces cyp1A2 and other liver specific proteins • Acts through inhibition of ras signaling

21. Catechins • Structurally related family of isomers and epimers • Known interaction with aryl hydrocarbon receptor • System distinguishes between the epimers catechin gallate and epicatechin gallate

23. ECG is Synergistic with MeChol In the presence of 1µM methylcholanthrene, ECG cooperatively induces cyp1A

24. Usnic Acid “Usnic acid will be the weight loss supplement of the new millenium.” James Shoemaker, MD “This is a young woman who almost lost her life. The fact that you can get these things over the internet is mind boggling.” Ron Busuttil, MD Recommended daily dose is 1g.

25. PREDICTIVTox - Predictive Hepatotoxicity Database • 50,000 Compounds screened in seven assays, two concentrations • Anchored to 5,000 literature accessible compounds • Only coupled predictive/experimental system

26. PREDICTIVTox - Predictive Hepatotoxicity Database • Built on Assay Explorer and ChemBioAE • Rapid visualization of data • Correlation of structural information with biological data • Allows user to quickly find and examine structural correlations

27. Typical Example - cyp1A induction assay • 1,280 compounds, quadruplicate, plus controls, two concentrations • 144 X 96 well plates • Biotek fluorescent plate reader • Approximately 13,000 assays

29. Sort and rank results against structure Mechanistic and Structural Analogies

30. Sulindac Induces cyp3A Activity

31. Effect of Kinase Inhibitors on cyp1A

32. Sulindac Induction is not Transcriptional

33. IRS1 PI3K Akt mTOR 4EBP Ras Raf MEK Erk mnk1 eIF4EP Translational Control Insulin Inhibition of PI3K shuts off translation Inhibition of ERK alters translational selectivity eIF4E

34. Aristolochic Acids Aristolochic Acid Aristolactam Aristolic Acid Aristolochic acids are carcinogens and are implicated in Chinese Herb nephropathy. They are metabolized by cyp1A.

35. Aristolochic Acid Analogs Induce cyp1A100µM

36. Sort and rank results against structure Mechanistic and Structural Analogies

38. Structural Homologs to Aristolochic Acid Apomorphine Trequinsin Dihydrexidine A 77636

39. Structural Homologs Also Induce cyp1A15 compounds induced >20 fold at 10µM

40. Summary • Simple in vitro toxicology assays coupled to a metabolically active biological system yields easily interpretable structure toxicity relationships. • Results can be used to explore structural and mechanistic hypotheses. • Assay Explorer and ChemBioAE provide a sophisticated framework for data capture, visualization and interpretation. • A coupled database/experimental system can provide the information necessary to minimize toxicity while simultaneously maximizing therapeutic effect.

41. Houston is a nice place to live but…

42. Sometimes we get a little flooding Tropical Storm Allison, June 8, 2001

43. CTIVTox™ Human Liver Cell Systems