Insert presentation name here 4/26/2017 Comparison of HµREL Co-culture Hepatocyte Model to Conventional In Vitro Models for Intrinsic Clearance and Metabolite ID J. Matthew Hutzler, Ph.D. Director, In Vitro Metabolism HµREL Symposium (2015 ISSX Meeting)

In Vitro Metabolism and Scaling Predicting Clearance Common methodology to evaluate metabolic stability is to incubate test compound (0.1 to 1 µM, or <<Km) with liver microsomes or hepatocytes and measure rate of substrate depletion (half-life) Clint = 0.693 x 25.7 g liver x mL inc x 45 mg mic protein = mL/min/kg t1/2 (min) kg body Wt mg mic protein g Liver Well-stirred model CLh = Qh x fu x CLint Qh + (fu*CLint) Clearance projection Slowly metabolized (i.e. low-turnover) drug molecules are becoming increasingly common For low-turnover compounds, traditional incubation systems (e.g. microsomes and hepatocyte suspensions) are inadequate for a reliable clearance projection due to incubation time limitations (~1-2 hr for microsomes and 4 hr for hepatocytes) Hutzler et al. (2015) Drug Metab Dispos. 43(12)

Cryopreserved Human Hepatocyte Suspension Literature Data Demonstrating Rapid Loss in Activity Effect of time (in humidified incubator at 37 C) on activities in cryopreserved hepatocyte suspensions Mean IT50 values (time where 50% loss in activity was observed) CYP1A2: 2.69 hrs CYP2C9: 4.47 hrs CYP2D6: 3.03 hrs CYP3A: 1.62 hrs UGT: 1.39 hrs Rapid loss of activity that is enzyme-dependent Incubation time limit: ~4 hrs Smith et al. (2012) J Pharm Sci. 101(10), 3989-4002

In Vitro Assay Option for Longer Duration Incubations HµREL® Hepatic Co-culture Models HµREL® - a hepatic co-culture platform (with stromal cells) Robust enzyme activity for days to weeks Plates arrive ready to use same day Multiple plate formats available (12, 24, 48, 96 well) Human, Rat, Dog, Monkey available on same plate for cross-species comparisons Can use any plateable lot of hepatocytes Q2 Solutions has conducted pilot studies with HµREL® co-culture system in effort to evaluate and potentially utilize for clients with low turnover compounds needing either clearance projection, or metabolite profile information Leslie Benet, Presentation at The Boston Society, July 2013

Low Clearance In Vitro Assays: A Comparison Study Study Design Summary Condition Suspension (4 hr) Plated Monolayer (24 hr) HµREL® (72 hr) Hepatocyte Lot LiverPoolTM 5-Donor Mixed Gender Cryoplatable Lot YMD (BioreclamationIVT) Thawing Media CHRM HµREL thaw Plating Media - Hu Hep Plating Media HµREL plate Incubation Media HMM HµREL Dosing Media Seeding density (cells/well) 50,000 45,000 30,000 Inc Volume 200 µL 100 µL Substrate Concentration 1 µM Plate Design 96 well Shaker speed 600 rpm 120 rpm Time Points (6) 0, 15 min, 0.5, 1, 2, 4 hr 0, 4, 6, 8, 18, 24 hr 0.25, 3, 6, 24, 48, 72 hr General procedures Thaw cells, incubate in suspension for total of 4 hrs Plate at 8am, dose at noon (t0), sample at 4 pm, 6 pm, 8 pm, 6 am, and noon next day Receive plates on a Tuesday morning, dose in afternoon, 72 hrs is Friday afternoon Stromal cell only plate included as a control for HµREL system study

Low Clearance In Vitro Assays: A Comparison Study Selected Substrates Compound Enzymes Involved in Metabolism ± Warfarin CYP2C9, CYP3A, CYP1A2 Timolol CYP2D6 Diazepam CYP3A, CYP2C19 Tolbutamide CYP2C9 Theophylline CYP1A2 Metoprolol Efavirenz CYP2B6 Verapamil CYP2D6, CYP3A, UGT Diclofenac CYP2C9, UGT Alprazolam CYP3A Glimepiride Meloxicam Prednisolone 13 substrates selected based on range of reported clearance values, as well as diversity in cytochrome P450 metabolic clearance mechanisms Minimal renal clearance Clearance rates estimated by substrate depletion methodology Scale-up to intrinsic clearance

Calculations of Intrinsic Clearance kdep = slope of time (min) vs. Ln% Remaining; 𝑘 𝑑𝑒𝑝 = 𝐿𝑛(2) 𝑡 1/2 In vitro half-life: In Vitro Intrinsic Clearance: Intrinsic Clearance (Clint): Clint in vivo back-calculated from Clin vivo using the well-stirred model 𝐶𝑙 𝑖𝑛𝑡,𝑖𝑛 𝑣𝑖𝑣𝑜 = 𝐶𝐿 𝑖𝑛 𝑣𝑖𝑣𝑜 𝑓 𝑢𝐵 𝑥 1− 𝐶𝐿 𝑖𝑛 𝑣𝑖𝑣𝑜 𝑄

Representative Metabolic Stability Figures Suspension NC NC NC Plated NC NC NC NC * 45% dep 70% dep NC HµREL® 77% dep *internal standard issue NC = no clearance

Representative Metabolic Stability Figures Suspension NC Plated HµREL® 88% dep 91% dep 96% dep NC = no clearance

Intrinsic Clearance Comparison (mL/min/kg) Hepatocyte suspension assay yields higher turnover than either plated format for moderate to high clearance compounds (consistent with literature) Clearance rate in HµREL co-culture system 2- to 7-fold higher than hepatocyte monoculture format, with better ability to measure clearance rate (11/13 compounds, or 85%)

In Vitro-In Vivo Correlation Analysis Suspended hepatocyte data correlates best overall for moderate/high clearance, but incubation time limits measuring clearance for low-turnover compounds Plated monoculture performs poorly (5/9 non-detects), and 24 hour incubation may not be long enough for a low-turnover compound HµREL co-culture trends toward under-prediction for moderate/high clearance compounds, but preferred over monoculture system Additional studies with more low-turnover compounds under way tolbutamide warfarin prednisolone Drop by Poster P10 to discuss more…

Metabolite Identification Guidance Expectations around Metabolite Profiling (FDA/EMA/Japan): Are there “major” circulating human metabolites, and are they covered in the toxicity species - MIST Are there “major” circulating human metabolites that may elicit clinically relevant drug interactions – DDI What are the primary metabolic clearance pathways, including the enzymes responsible – DDI Prior to Phase I dosing, in vitro systems are relied upon to predict human metabolites To support selection of toxicity species To predict the potential for victim-based DDIs and/or PK variability in the clinic For moderate/high clearance drugs, traditional in vitro systems are used for these assessments For low clearance drugs, HLMs or suspended hepatocytes are often inadequate at investigating metabolic pathways Study Design 4 hour suspension hepatocyte incubation 24-well plate design (HµREL), with ~188K hepatocytes/well 4, 24, 72, and 168 hr incubations (no media change) 10 µM meloxicam, timolol, and XK-469 Analysis using high-resolution mass spectrometry (Thermo Orbi-TRAP) Comparison of metabolite profiles

Meloxicam Metabolism 10 15 20 25 30 35 Time (min) Melox-OH Melox-COOH HµREL – 4 hr HµREL – 24 hr HµREL – 72 hr HµREL – 168 hr Standard Hepatocyte 4 hr Incubation Meloxicam Melox + 4H + O + Melox + 2 H2O Suspended hepatocytes: Observed only trace levels of +O (Melox-OH) HµREL Co-culture: Observed progression from +O (Melox-OH) to subsequent acid (Melox-COOH) over time Robust metabolism to yield multiple metabolites Comparison to in vivo data: Based on 14C-human: Melox-OH ~9% dose Melox-COOH ~60% dose Turck et. al. 1996 Conclusions: Melox-COOH and Melox-OH readily observed\ Better correlation with in vivo met ID data +4H+O and +2H2O peak significant in HµREL, in vivo relevance unknown

Insert presentation name here Timolol Metabolism 4/26/2017 10 15 20 25 30 Time (min) Timolol Timolol + Gluc + O + 2O Morpholine ring open +2O - C2H2 HµREL – 4 hr HµREL – 24 hr HµREL – 72 hr HµREL – 168 hr Standard Hepatocyte 4 hr Incubation Suspended hepatocytes: Observed only trace levels of metabolites, largest +O HµREL Co-culture: Seeing progression from +O to morpholine ring opening (+2O) to loss of ethyl (-C2H2) Seeing robust metabolism to yield multiple metabolites Comparison to in vivo data: Based on 14C-human: Morpholine ring open (+2O) ~22% dose Loss of ethyl bridge from morpholine ~7% dose Tocco et. al. 1980 Conclusions: Morpholine ring opening with subsequent loss of ethyl bridge readily observed Intriguing that +gluc was not observed in suspensions but significant in HµREL Loss of ethyl bridge

XK-469 Metabolism 10 15 20 25 30 35 Time (min) HµREL – 24 hr + O (AO) + Glycine + Taurine + AcetylCys – 2H + Cys – 2H Standard Hepatocyte 4 hr Incubation Suspended hepatocytes: Observed only trace levels metabolites, largest +O HµREL Co-culture: Seeing +O associated with aldehyde oxidase as largest peak Seeing robust metabolism to an array of non-P450 metabolites Comparison to in vivo data: Based on human data: AO metabolite accounts for 54% of total urinary excretion, and is most predominant metabolite Anderson et. al. 2005 Conclusions: Clearly suggests non-P450 pathways such as AO are viable for 7 days A good example of the need for a reliable in vitro system for prediction of metabolic pathways in human

Acknowledgements Clearance Studies Mark VandenBranden Todd Hieronymus Andy Staton Barbara Ring HµREL Metabolite ID Studies Eric Novik Matt Shipton Taysir Chamem Todd Hieronymus David Heim Rich Burton Shelby Anderson Xiusheng Miao

Back-Up Slides

Instrumentation/Method Utilized Mass Spectrometry System: Thermo LTQ Orbitrap XL Ionization: ESI+; Source: 5.0 kV; Capillary: 350゜C Fragmentation Method: HCD Collision Energy: 25 (meloxicam), 50 (timolol), 40 (XK469) Chromatography System: Shimadzu Nexera X2 UHPLC Column: Supelco Ascentis Express C18, 2.7 µm, 2.1×150mm; 25゜C Mobile Phase: A: 10 mM ammonium formate + 0.1% formic acid B: acetonitrile Flow Rate: 0.15 mL/min. Gradient: Company Confidential

Metabolic Stability Data Figures NC NC Suspension 51% dep 53% dep NC Hµrel® Only theophylline and alprazolam showed no clearance in Hurel® system NC = no clearance

In Vitro Metabolism and Scaling Extrapolation Guidelines A cut-off for half-life extrapolation using in vitro data needs to be established To confidently extrapolate an in vitro half-life, at least 20% depletion of substrate needs to be observed to be interpreted as metabolism (i.e. beyond biological/analytical noise) 20% depletion at t240 min roughly equates to a half-life ~3x the incubation time (~720 min), but all data points need to be considered An in vitro system enabling a longer incubation time would be beneficial to improve our confidence in prediction 104 98 91 80 Assuming reasonable extrapolation of half-life to ~3x beyond length of assay (extrapolate t1/2 to 720 min): Clint limit: ≤11.9 mL/min/kg Clh limit: ≤7.5 mL/min/kg (36% LBF) Human liver blood flow (LBF): 20.7 mL/min/kg t1/2 = 630 min (2.6x > inc time)

Low Clearance In Vitro Assays Options and Pros/Cons Relay Method Using Cryopreserved Hepatocytes – Relay every 4 hours Pros Cons Utilizes standard max 4 hr incubation time (literature support) Uses 4-5x the amount of hepatocytes (~$3000 in hepatocytes) Can use any lot of pooled cryopreserved hepatocytes Labor intensive (4-5 days), depending on number of relays Good correlation with human in vivo data (demonstrated IVIVC) Have to account for lost compound during relays (calculation) IVIVC also demonstrated for pre-clinical species (rat/dog) Dilution factor for each relay Number or relays flexible (max of 5) Challenge to reproduce results (personal communication) *assuming ~$600 per vial Thaw at T=0 hrs Thaw at T=4 hrs Thaw at T=16 hrs Thaw at T=12 hrs Thaw at T=8 hrs * Relay method also shown to be suitable to generate metabolites for met ID Di et al. (2012) Drug Metab Dispos. 40, 1860-1865 Di et al. (2013) Drug Metab Dispos. 41, 2018-2023 *Ballard et al. (2014) Drug Metab Dispos. 42, 899-902

Low Clearance In Vitro Assays Options and Pros/Cons Plateable Cryopreserved Human Hepatocytes – single donor or plateable pool Pros Cons Enzyme activity more stable compared to suspension Enzyme activity decreases over first 24 hrs (~50%) Practical solution – plate cells and dose 4 hrs later Not all lots of hepatocytes are plateable Some plateable pools now available (5 or 10 donors) There is question about the plating efficiency of multiple donors; single donor, enzyme variability a concern (no PMs) 24 hrs incubation limit may not be long enough for low CL * Hepatocytes were plated and allowed to attach for 4 hours prior to initiation of study Effects on specific activities in plated cryopreserved hepatocytes (4 preparations) IT50 estimates for CYP1A2 and CYP3A: 21.3 ± 2.1 and 28.8 ± 20.4 hrs Smith et al. (2012) J Pharm Sci. 101(10), 3989-4002

Metabolic Stability Data Figures Suspension Plated Hµrel® Verapamil 1-3 all different plates; verapamil 3 run on different date