1. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 An Integrated Approach to More Accurately Assess Cholestatic Liability of Drugs Mark S. Warren 20 th North American ISSX meeting | Orlando, FL | Oct 20, 2015

2. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 Drug-Induced Liver Injury (DILI) Common reason for failure of pharmaceuticals during drug development, common reason for removing approved medications from the market Interfering with normal bile flow can result in mild & transient cholestasis… or severe hepatocellular damage Often difficult to predict 2

3. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 The effect of drugs on bile salt clearance in hepatocytes Bile Salts: Clearance is heavily dependent on transporters. Drug induced inhibition of transport -> cholestasis. Increased hepatic accumulation -> hepatocellular damage. Drugs & Metabolites: Clearance is heavily dependent on transporters BSEP binding site is inside cells – intracellular concentration (not plasma concentration) is important. Non-bile salt transporters also involved in mediating drug concentrations. 3

4. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 Understanding Cholestatic Liability of Drugs We have combined two approaches to better understand potential DILI: Mechanistic transporter models: MDCK-II cells transfected with various combinations of transporters involved in hepatic clearance of drugs and bile salts (NTCP, OATPs, BSEP, P-gp, etc.) Hepatocyte co-culture models 4

5. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 Contribution of Individual Transporters to Taurocholate Transport 5 Transcellular Transport Intracellular Accumulation

6. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 Contribution of Individual Transporters to Taurocholate Transport 6 Transcellular Transport Intracellular Accumulation Apical Efflux

7. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 Mechanistic study of drugs with DILI liability 7 Effect on TCA secretion Effect on Cellular TCA retention Effect on Apical TCA efflux

8. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 Mechanistic study of drugs with DILI liability 8 Effect on TCA secretion Effect on Cellular TCA retention Effect on Apical TCA efflux

9. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 Rifampicin BDDCS Class I, C max ~10µM, fu ~20% Extensive metabolized in liver to Desacetyl-Rifampicin Clearance: hepatic ~70%, renal: ~30 (~7% parent) High biliary concentrations of DES-RIF and RIF Rifampicin is known to cause liver injury (~1% of patients), incl. fatal liver failure A potent inhibitor of OATPs, MRPs and BSEP Does not inhibit NTCP (up to 300µM) or ASBT* PXR activator Induce metabolic enzymes Up-regulate OATPs, MRPs and P-gp 9 Rifampicin 25-desacetyl rifampicin

10. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 Rifampicin is not transported by NTCP, BSEP, OATP1B1 Mechanistic study using clearly defined MDCK models expressing various transporters revealed that Rifampicin is not transported by NTCP or BSEP 10 Transcellular Transport Intracellular Accumulation Apical Efflux

11. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 Rifampicin is a substrate of P-gp Mechanistic study using clearly defined MDCK models expressing various transporters revealed that Rifampicin is a substrate of P-gp, but is not transported by NTCP nor BSEP, and its intracellular concentration heavily depends on P-gp 11 Transcellular Transport Intracellular Accumulation Apical Efflux

12. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 Effect of P-gp on Rifampicin and Metabolite P-gp dramatically reduces the intracellular concentrations of Rif and Des-Rif in cells expressing P-gp. P-gp may be a key determinant of the hepatic concentration and liver toxicity of Rif and Des-Rif. 12

13. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 P-gp protects against rifampicin hepatotoxicity 13  P-gp decreases intracellular concentrations of potentially hepatotoxic agents…  This suggests that P-gp inhibitors may potentiate hepatotoxic BSEP inhibition.

14. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 Potential Synergistic Effects of RIF Co-Meds on Blocking Hepatic Bile Salt Clearance? The incidence of liver injury in patients taking Pyrazinamide/Rifampicin is ~4 times higher than taking Rifampicin alone (Yew & Leung 2006) Protease inhibitors (Lopinavir/Ritonavir, Saquinavir/Ritonavir) caused unexpected liver injury in HIV infected TB patients (Nijland et. al. 2008, Schmitt et. al. 2009) 14 Yew WW, Leung CC. Antituberculosis drugs and hepatotoxicity. Respirology 2006; 11(6):699-707 Nijland HM et al. High incidence of adverse events in healthy volunteers receiving rifampicin and adjusted doses of lopinavir/ritonavir tablets. AIDS. 2008;22(8):931–935 Schmitt C et al. Unexpected hepatotoxicity of rifampin and saquinavir/ritonavir in healthy male volunteers. Archives of Drug Information. 2009;2(1):8–16.

15. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 Protease Inhibitors Inhibit P-gp and Aggravate Rifampicin Toxicity Ritonavir + Lopinavir cause an increase in intracellular concentrations of Rif and Des- Rif in MDCK models by inhibition of P-gp. These results suggest that if co-administrated with Rif, they may aggravate Rif hepatotoxicity. 15

16. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 Methods: Hepatocyte Co-Cultures 16 Culture SystemHurel Hepatic Co-Cultures Lot/SpeciesHuman (Hurel H1004) Test CompoundsCyclosporin A (0-50 µM), Ritonavir (0-200 µM) Troglitazone (0-629 µM), Bosentan (0-199 µM) Erythromycin Estolate (0-250 µM) Bile Acids Mix (40x)52.8 µM glycochenodeoxycholic acid 15.6 µM chenodeoxycholic acid 15.2 µM glycodeoxycholic acid 16 µM deoxycholic acid 14 µM glycocholic acid Incubation Time1 hr (compound incubation), 24hr, 48hr Toxicity AssaysCellTiter-Blue, CellTiter-Glo Albumin Assay (Liver-Specific Function) Replican = 2

17. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 Albumin Assay for Liver-Specific Function Albumin secretion was measured in Hurel hepatic co- culture models. Bile salts alone have no effect Hepatotoxic drugs decrease albumin secretion in this assay. Albumin is a good marker for hepatotoxicity 17

18. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 Cyclosporine A Hepatotoxicity is Potentiated by Bile Salts Albumin secretion was measured in the presence of Cyclosporine A with and without bile salts. After 24 hours, increased hepatotoxicity is seen in the presence of drug + bile salts compared to drug alone. 18

19. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 Ritonavir Hepatotoxicity is also Potentiated by Bile Salts Albumin secretion was measured in the presence of ritonavir with and without bile salts. After 24 hours, increased hepatotoxicity is seen in the presence of drug + bile salts compared to drug alone. 19

20. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 Hepatotoxicity of Bosentan For compounds like bosentan, the toxicity can take up to 48 hours to present Toxicity is enhanced in the presence of bile salts. 20

21. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 Rifampicin in Hepatic Co-Cultures Hepatotoxicity of rifampicin was measured with bile salts +/- ritonavir. Rifampicin hepatotoxicity is greatly enhanced in the presence of ritonavir. 21

22. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 Summary The clearance of bile salts is heavily dependent on transporters. Numerous transporters are involved, directly or indirectly. Drug induced inhibition of bile salt uptake and/or efflux can cause blockage of bile salt hepatic clearance (resulting in cholestasis), and potentially result in increased hepatic bile salt accumulation (resulting in hepatocellular damage). Mechanistic transporter models can show potential cholestatic risk of drugs, while hepatic co-cultures can confirm potential hepatotoxicity. 22

23. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 Conclusions Several drugs (cyclosporine, rifampicin, bosentan) showed increased hepatotoxicity in the presence of bile salts, suggesting that blocking bile salt clearance via inhibiting bile salt transporters likely attributes to their liver toxicity. Using mechanistic transporter models, rifampicin (and desacetyl-rifampicin) potently inhibit BSEP but not NTCP, while their intracellular concentrations are mediated by P-gp. Therefore, P-gp inhibition by a co-medication can increase hepatotoxicity. The hepatocyte co-culture model demonstrates that ritonavir potentiates rifampicin hepatotoxicity in a bile salt dependent manner. These results may provide a mechanistic explanation on the high incidence of unexpected liver injuries in HIV-infected TB patients. Well-defined transporter models are useful tools for mechanistic studies of bile salt transport and its inhibition by drugs, while the resultant hepatotoxicity can be confirmed with the hepatocyte co-culture model. This combination allows for the ability to run detailed mechanistic transporter studies that can identify potential transporter interactions and confirm hepatotoxicity. 23

24. www.optiviabio.com | info@optiviabio.com | +1.650.324.3177 Thank you! 24 Eric Novik Cheul Cho Eric Pludwinski Matt Shipton Yong Huang Jason Baik Dawn Stricker