1. Y OUNG I NNOVATORS 2009 Hepatobiliary disposition of troglitazone and metabolites in sandwich-cultured hepatocytes: Integration of pharmacokinetic modeling and Monte Carlo simulations Jin Kyung Lee, Ph.D. Qualyst, Inc

2. I NTRODUCTION Young Innovators 2009 Troglitazone (TGZ), a peroxisome proliferator-activated receptor gamma (PPAR  ) agonist, was approved as an insulin-sensitizing drug for the treatment of non-insulin-dependent diabetes mellitus. TGZ was withdrawn from the market in 2000 due to reported cases of severe idiosyncratic liver failure.

3. OBJECTIVE This study was designed to assess the capability of sandwich-cultured hepatocytes coupled with modeling/simulation to predict the impact of altered transport function on hepatic accumulation of drugs/metabolites. Young Innovators 2009

4. M ATERIALS AND M ETHODS TGZ (10µM) was added to rat SCH (day 4) and human SCH (days 6-7). Samples were collected from medium, cells+bile, and cells from rat SCH at 10, 20, 30, 60, 90 and 120 min, and from human SCH at 30, 60 and 120 min. Young Innovators 2009 [with Ca 2+ ; cells+bile][without Ca 2+ ; cells] = [Bile]

5. M ATERIALS AND M ETHODS Differential equations describing substrate flux in each compartment were fit simultaneously to the data by nonlinear least-squares regression Parameter estimates with associated variance and uncontrolled error served as input for Monte Carlo simulations. Young Innovators 2009 [Ka] [Kb] [Kc] [y1] [y2][y3] Compartmental Modeling Monte Carlo Simulation

6. R ESULTS Young Innovators 2009 Consistent with in vivo data, TGZ ( ● ) was extensively metabolized to TS ( ● ), and to a lesser extent to TG ( ● ) and TQ ( ● ) in rat SCH; the biliary excretion of TS was greater than that of TG in rat SCH. MediumHepatocytes Bile

7. R ESULTS Young Innovators 2009 Hepatocyte accumulation of TS was extensive, with intracellular concentrations reaching 132-222 µM based on data generated in rat SCH; intracellular TGZ concentrations ranged from 7.22-47.7 µM. In human SCH, intracellular TS and TGZ concentrations ranged from 136-160 µM and 49.4-84.7 µM, respectively (data not shown). TGZ-mediated Bsep inhibition (IC 50 = 3.9 µM) TS-mediated Bsep inhibition (IC 50 = 0.4-0.6 µM) V

8. R ESULTS A compartment model was developed to describe the hepatobiliary disposition of TGZ and its metabolites in medium, cell and bile To explore the hepatobiliary disposition of TS in response to impaired hepatic transport, simulations were performed by modulating K bile,TS Young Innovators 2009 ParametersEstimatesCV% K uptake, TGZ 0.007 min -1 2 K uptake, TS 0.004 min -1 36 K uptake, TG 0.001 min -1 388 K f, TS 0.383 min -1 10 K f, TG 0.061 min -1 11 K f, TQ 0.001 min -1 17 K other 0.064 min -1 23 K efflux, TS 0.018 min -1 39 K efflux, TG 0.135 min -1 34 K bile, TS 0.035 min -1 21 K bile, TG 0.087 min -1 48 K flux, TS 0.255 min -1 24 K flux, TG 0.137 min -1 57 T flux 16.7 min8

9. R ESULTS Cellular accumulation of TS was sensitive to impaired or increased K bile,TS, but changes in K bile,TS were not reflected in TS accumulation in medium. Cellular concentrations of TS could reach >1200 µM if TS biliary excretion was impaired 10-fold based on simulations. In contrast, medium concentrations of TS could increase up to 4.8 µM when TS biliary excretion was decreased 10-fold. Young Innovators 2009 HepatocytesMedium

10. D ISCUSSION Data from sandwich-cultured hepatocytes were consistent with TGZ disposition previously reported in vivo in rats (TS > TG). Intracellular concentrations of TGZ and TS were much higher than the previously reported IC50 of TGZ and TS for Bsep- mediated taurocholate transport in isolated canalicular rat liver plasma membranes (Funk et al., 2001). Young Innovators 2009

11. D ISCUSSION In clinical studies, plasma concentrations are often used to examine the effect of canalicular transport protein modulation. Simulations suggest that plasma concentrations may not always reflect changes in hepatic exposure when biliary excretion pathways are modulated. Young Innovators 2009

12. C ONCLUSION Data obtained from sandwich-cultured hepatocytes coupled with pharmacokinetic modeling and Monte Carlo simulation is capable of predicting the impact of altered transport function on hepatic (reflected in the accumulation in hepatocytes) and systemic (reflected in the accumulation in medium) exposure. Young Innovators 2009

13. A CKNOWLEDGMENTS Kim Brouwer, Pharm.D, Ph.D Gary Pollack, Ph.D David Miller, Ph.D Elaine Leslie, Ph.D Mary Paine, Ph.D Nita Patel, Ph.D Young Innovators 2009

14. R EFERENCES Graham DJ, Green L, Senior JR and Nourjah P (2003) Am J Med 114:299-306. Funk C, Pantze M, Jehle L, Ponelle C, Scheuermann G, Lazendic M and Gasser R (2001) Toxicology 167:83-98. Loi CM, Young M, Randinitis E, Vassos A and Koup JR (1999). Clin Pharmacokinet 37:91-104. Izumi T, Enomoto S, Hosiyama K, Sasahara K, Shibukawa A, Nakagawa T and Sugiyama Y (1996) J Pharmacol Exp Ther ka277:1630-1641. Liu X, LeCluyse EL, Brouwer KR, Gan LS, Lemasters JJ, Stieger B, Meier PJ and Brouwer KLR (1999) Am J Physiol 277:G12-21. Young Innovators 2009

15. BIOS/C ONTACT INFO Jin Kyung Lee, Ph.D. Senior Scientist Qualyst, Inc. 2810 Meridian Parkway, Suite 100, Durham, NC 27713 E-mail: jinlee@qualyst.com Young Innovators 2009