Prospective Liver Safety Comparison of Two Treatments for Autosomal-Dominant Polycystic Kidney Disease (ADPKD) Using Quantitative Systems Toxicology Modeling Woodhead, J.L.*, Pellegrini, L.#, Siler, S.Q. *, Shoda, L.K.M. *, Watkins, P.B. *, Howell, B.A. * *DILIsym Services, Inc., a Simulations Plus Company, Research Triangle Park, NC, USA; #Palladio Biosciences, Inc., Newtown, PA

ABSTRACT RESULTS Objectives: Lixivaptan, a -2 receptor antagonist, is in development for the treatment of ADPKD, an orphan disease In Vitro Results PBPK Representation and Custom SimPops with high unmet medical need. The main objective of this research was to The PBPK representation for lixivaptan prospectively compare the potential for and its major metabolites was lixivaptan to cause liver toxicity to a constructed using clinical data for comparator drug in the same class, optimization and validation. The clinical , which has produced off-target trials used were Phase I trial results (n = liver signals in clinical trials1. 67) for seven-day 100 mg BID and 400 mg BID dosing regimens. The simulation Methods: In vitro data relating to reactive results reasonably matched the data. oxygen species formation, mitochondrial Liver accumulation data from a rat toxicity, and bile acid transporter inhibition WBAR study and from in vitro studies for lixivaptan and its metabolites were collected for this work were consistent collected. Using these data, lixivaptan and with simulated liver concentrations. its metabolites were represented in DILIsym, The exposure range of the a platform QST model of drug-induced liver In vitro experiments customized SimPops was validated injury. Lixivaptan PBPK was also measured mitochondrial against the observed exposure represented within DILIsym, incorporating dysfunction, ROS generation, range from the clinical trial data for clinical trial PK data. Proposed ADPKD and bile acid transporter all four chemical species (lixivaptan treatment dosing regimens were simulated inhibition due to all four is shown at right). The range of and the predicted potential for liver enzyme chemical species. For the simulated exposures aligned well elevations was compared to that previously mitochondrial assay and the with the observed data; outliers 2 determined for tolvaptan in DILIsym . ROS assay, a parallel culture were expected based on the fact was run in order to assess that the simulated population was Results: Lixivaptan was not predicted to the intracellular concentration larger than the population in the cause liver enzyme elevations in a simulated present in each assay; the clinical trial. human population which includes variability assay results were corrected in toxicity susceptibility and for these concentrations pharmacokinetics, while tolvaptan was before the parameter values Simulation Results correctly predicted to cause rare liver were calculated. enzyme elevations in a similar population Clinical Simulated Dose and Parameter (Table 1). Mechanistic simulations at Drug ALT > 3X ALT > 3X Calculated Parameter Comparison Duration Settings supratherapeutic doses suggest that ULN ULN potential liver toxicity mechanisms for Study not Value**** 200/100 mg Default lixivaptan are different from those identified DILIsym Lixivaptan yet 0/285 Mechanism Unit QD, 12 wks measured# for tolvaptan. Parameter Lixivaptan WAY-138451 WAY-141624 WAY-138758 Tolvaptan** conducted Coefficient Mitochondrial Conclusions: Lixivaptan was predicted to for ETC µM 535 250 N/A N/A 729 90/30 mg Default 18/229 Dysfunction Tolvaptan^ 4.4% be safer than tolvaptan with respect to the inhibition QD, 24 wks measured# (7.86%) liver toxicity mechanisms represented in RNS/ROS DILIsym. Quantitative and qualitative Oxidative production -4 -3 Simulated Simulations predicted lixivaptan to be mL/nmol/hr 5.45 x 10 2.12 x 10 N/A N/A N/A Dose and Parameter Clinical ALT > differences were identified between the two Stress rate ALT > 3X Duration Settings 3X ULN less toxic than tolvaptan; while tolvaptan drugs. These findings pave the way for constant ULN had significant ALT elevations in its confirmatory clinical trials with lixivaptan in On treatment BSEP 100 mg BID Default SimPops simulation, lixivaptan had only ADPKD. similar to 0/285 inhibition µM 15* 8.6* 39.5* 5.6* 10*** for 60 days measured sub-clinical ALT elevations. Simulations constant placebo for lixivaptan suggested a low rate of 200 / 100 Clinical study INTRODUCTION Default ALT elevations at 400 mg BID, which mg for 12 not yet 0/285 Bile Acid NTCP measured suggests that the simulation results may • Lixivaptan is a V2 Transporter inhibition µM 19* N/A 85.8* 8.9* N/A weeks conducted be slightly conservative. ROS was found antagonist that is under investigation for Inhibition constant 400 mg BID Default 0/67 7/285 the treatment of autosomal-dominant for 7 days measured to be the main mechanism responsible polycystic kidney disease (ADPKD), an Basolateral for simulated ALT elevations at the Dose and Simulated ALT > inhibition µM 70* 54* 16.3* 4* N/A Parameter Settings supratherapeutic dose, in contrast with inherited orphan disease characterized Duration 3X ULN constant** the case of tolvaptan in which bile acid by progressive kidney failure. 400 mg BID, 7 Default measured 7/285 accumulation and ETC inhibition were days • Lixivaptan is in the same class of drugs * Values are IC values; mode of inhibition was not measured in vitro. In a 2 50 400 mg BID, 7 No parent- found to be the mechanisms of toxicity . as tolvaptan, an investigational ADPKD sensitivity analysis, the worst-case inhibition scenario (noncompetitive BSEP and 0/285 days generated ROS ^ Tolvaptan simulation results are from Woodhead 20162. treatment that caused liver toxicity in a basolateral inhibition, competitive NTCP inhibition) was assumed; toxicity results Phase III clinical trial. were unaffected. As a result, mode of inhibition was determined to not affect the

• In previous work, DILIsym was simulation and Ki investigation studies were not commissioned. Exposure-Toxicity Relationship employed to model tolvaptan-mediated liver injury; DILIsym was able to ** Tolvaptan parameters are taken from in vitro experiments undertaken for this The simulation of lixivaptan at the Expected recapitulate the observed toxicity, research. Previously published DILIsym parameters are available in Woodhead et clinical supratherapeutic dose of 400 mg BID shows implicating a combination of bile acid al., Tox. Sci. 20162. The published ETC inhibition parameter was 1030 mM, which exposure a distinct relationship between exposure and transporter inhibition and mitochondrial is not significantly different from the measured value here. range ALT elevations. From this relationship, it is electron transport chain (ETC) inhibition apparent that the expected exposure range for the 200/100 mg split daily dose proposed as responsible for the toxicity. *** IC50 value for tolvaptan was measured for this research. A Ki value was • A DILIsym representation was thus measured for the previously published tolvaptan work; the published value is for use in the clinic is well below that which produces clinically significant (>3X ULN) ALT constructed for lixivaptan and its three somewhat higher than the value reported here. However, personal communication Predicted exposure range elevations in the simulations. This also major metabolites, WAY-138451, WAY- with the experimentalists suggested that the initial IC50 value calculated in that for clinically stands in contrast with tolvaptan, where no 141624, and WAY-138758, in order to experiment was not substantially different from that measured here. relevant ALT compare the potential for lixivaptan to elevations exposure-response relationship was 1 cause hepatotoxicity with that simulated **** Comparisons of parameter values should be undertaken with caution, as they observed in the clinic and where simulations for tolvaptan. must be placed in context with exposure for their full usefulness. suggest that exposure-related parameters are not risk factors for toxicity2. METHODS CONCLUSION Simulations predicted lixivaptan to be less • In vitro toxicity data were collected in • A PBPK representation for lixivaptan likely than tolvaptan to cause liver injury in order to determine the effect of and its metabolites was constructed Vesicle BA Transporter Assay clinical trials for ADPKD. Furthermore, lixivaptan and its three major within DILIsym using plasma time mechanistic differences between lixivaptan metabolites on oxidative stress course data for all four chemical species and tolvaptan were identified, suggesting that generation, bile acid transporter as well as in vitro and in vivo liver it would be even more unlikely for lixivaptan inhibition, and mitochondrial accumulation data. to replicate tolvaptan’s negative clinical dysfunction. Lixivaptan and WAY- • A customized SimPops was created to experience. The simulations therefore support 138451 had an effect on all three charaterize lixivaptan exposure the continued development of lixivaptan for mechanisms; WAY-141624 and WAY- variability. This SimPops was based on ADPKD treatment. This research 138758 were bile acid transporter the v4A_1 SimPops included in DILIsym Seahorse Mitochondrial Assay demonstrates the potential for using QST inhibitors but did not induce oxidative v6A which includes variability in techniques to prospectively compare stress or affect the mitochondria. parameters related to each of the molecules in the same class for toxic • DILIsym parameter values were toxicity mechanisms represented in potential in order to select the molecule that DILIsym. A SimPops with variability in Clinical calculated using the in vitro data for Predictions is most likely to succeed. each of the mechanisms for which an exposure-related parameters was

effect was measured. Calculated IC50 created and superimposed upon the ACKNOWLEDGEMENTS v4A_1 SimPops. values were used as Ki values for • Palladio Biosciences, Inc. inputs; mitochondrial dysfunction was Schematic of the workflow for the DILIsym analysis of lixivaptan. • Clinical trial protocols and proposed • The members of the DILI-sim Initiative modeled in MITOsym, and oxidative protocols for lixivaptan were simulated stress was modeled in DILIsym. and compared to published simulation results for tolvaptan. References available upon request