Introduction to Hurel ISSX H µ REL ® C ORPORATION

 Co-cultured hepatic models: primary cryopreserved hepatocytes cultured together with cells of a non-parenchymal, stromal type.  5 species Hμ REL human™ Hμ REL humanPool™  7 ModelsHμ REL rat™ Hμ REL ratWH™ Hμ REL dog™ Hμ REL minipig™ Hμ REL primate™  Focus on practicality and convenience as well as function.  plates delivered by air overnight.  ready for use after brief acclimation period.  contract research services also available. Hµ REL ® Overview 2

HµRELhuman ™ HµRELdog ™ HµRELrat ™ HµRELprimate ™ Hµ REL ® delivers stable, enduring hepatic competency 3 High-throughput (384-well) & Larger Formats

Identifying a window of stable enzymatic activity (Hμ REL human ™ ) By day 7 the cells have finished remodeling and stabilized function. Day 7 of culture will be defined as “Customer Day 1” and the first day of the window of stability. The window will continue for 18 days. Metabolite formation rates will be normalized to Customer Day 1 for easier inter-lot comparison. Two weeks of stable culture is a long enough window of time for most Tox and DMPK applications. Culture Day 4

Canaliculus formation co-cultures are shown on culture Day 31; canaliculi form starting on Day 6 5

7-day metabolite generation: Hµrelhuman™ and Hµreldog™ in vitro: in vivo: 6

in vitro: in vivo: 7-day metabolite generation: Hµrelhuman™ and Hµreldog™ 7

Time- and concentration-dependent inhibition: ketoconazole ( potent CYP 3A4 competitive inhibitor) Cells were dosed with ketoconazole at various concentrations for 72 hours. CYP3A4 function was monitored by 1-OH-midazolam formation. After 72 hours the inhibitor was removed and recovery was monitored for an additional 72 hours. Concentration-dependent inhibition is evident immediately. Full recovery of CYP function after 24 hours of recovery. 8

Time- and concentration-dependent inhibition: diltiazem (time-dependent CYP 3A4 inhibitor) Dosing and recovery schedules were identical to the ketoconazole protocol. Concentration dependent inhibition is not immediately evident. Time dependent inhibition is observed after dosing with compound for 72 hours. 9

Time- and concentration-dependent induction: rifampin (CYP 3A4 inducer through PXR activation) Dosing and recovery schedules were SIMILAR to the ketoconazole protocol. An additional recovery time point of 120 hours was added. 24 hours after compound is removed induction is even more pronounced. 72 hour recovery is concentration dependent. Full recovery by 120 hours after dosing. 10

Klatt et al (2011) Pharmaceutics Introducing: Hμrelflux™ Direct-Measurement Efflux Transport Assay 1 1 patent pending 11 European ISSX 2015 Poster - P150

Cells + Bile Cells Bile Cells Cells + Bile Hurel Method 1 Well 1 Well 2 Well 1 Direct Bile Measurement Indirect Bile Measurement 1 Patent Pending. 12 Biliary Excretion: Two assay methods

Taurocholic Acid—human hepatocytes Hurel MethodLiterature: Gel Overlay Culture / Indirect Measurement* Uptake Rate (pmol/min/mg protein) 38  (23-25, Life Tech.) Biliary Clearance (  l/min/mg protein) 23  Biliary Excretion Index (%) 66  * Bi et al (2006) Drug Metabolism and Disp. (BEI = 67%) Bile Cell Bile 13

Hurel MethodLiterature: Gel Overlay Culture / Indirect Measurement* Uptake Rate (pmol/min/mg protein) 2.0  ( , Life Tech) Biliary Clearance (  l/min/mg protein) 0.3  Biliary Excretion Index (%) 40  3 37 Estradiol-Glucuronide—human hepatocytes Bile * Bi et al (2006) Drug Metabolism and Disp. Cell (BEI = 40%) Bile Cell Bile 14

Biliary Excretion—Rat In Vitro and In Vivo Pharmacokinetics * Lundquist (2014) Drug Metabolism and Disp. In vitro CL bile Assumptions: Intrinsic Cl int, bile values were converted to ml/min/kg based on 200 mg protein/g liver and 40 g liver/kg (Seglen, 1976) SubstrateRat In Vitro PK (Hurel Method 1 ) Rat In Vitro PK (Hurel Method 1 ) Rat In Vivo PK *(Literature) Fold Difference (Hurel/Literatur e) Intrinsic CL int, biliary (µl/min/mg protein) Predicted CL biliary (ml/min/kg) In Vivo CL biliary (ml/min/kg) Digoxin 0.31    Rosuvastatin 3.5    Estradiol- Gluc 2.7   0.4 n/a Taurocholate 8.4   4.8 n/a Pravastatin 0.29   0.14 n/a 15

16 ● Significant decrease in TC50 values in Hµrel dog>rat>>human co-cultures following 7&14-days treatment with cyclophosphamide as compared to 24hr treatment Human Rat Dog Cyclophosphamide: Cytotoxicity following 24hr treatment as compared to 7&14 day treatment

Time-based toxicity signals correlated to toxicity mechanisms 17

Effect of CYP Inhibitor for Cyclophosphamide 18 CellTiter-Blue Assay Human Co-Culture After 5 day dosing Cell Index (ACEA)

Dual-Chamber Applications liver / heart liver / kidney liver / lung liver / disease model liver / transfected target 19 confidential

Tegafur: Tegafur (pro-drug) 5’-fluorouracil (active metabolite) Liver CYP Pro-drug that converts to 5-FU in the liver F Hµ REL flow™ multi-tissue proof of concept colon cancer n liver t t 20 confidential

Hµ REL flow™ POC demonstration: Tegafur efficacy requires recirculation through a functional liver compartment 21 confidential

o Highly stable, reproducible cellular competency Most cells per well vs. competition o Long endurance and high function enables repeat-dosing studies drug-drug interactions complete metabolite formation o Emphasis on lab practicality and convenience air-shipped throughout W. Europe and N. America plates arrive ready for use after brief acclimation all standard formats available all media is included Hμ REL Advantage 22 confidential