A Wholly-Owned Subsidiary of Frontage Laboratories, Inc. Novel BSEP and MDR3 Inhibition Assays for Investigating Drug- Induced Liver Injury Kan He, PhD Outline • BSEPcyte®: BSEP inhibition format • MDR3cyte®: MDR3 inhibition format Why BSEP • Physiologically important: BSEP is the primary mechanism for export of bile salts – The driving force for bile salt enterohepatic recirculation and bile flow – “Vacuum cleaner” to maintain low intracellular level of bile salts • Validated target with clinical phenotype: BSEP mutations lead to human liver diseases – Progressive Familial Intrahepatic Cholestasis 2 (K903X, Intron 4 (+3)A>C) • Liver failure, death – Benign Recurrent Intrahepatic Cholestasis type 2 (E297G, R432T, V444A) – Associated with intrahepatic cholestasis of pregnancy • BSEP inhibition associated with Drug induced liver injury (DILI) DILI accounts for >50% acute liver failures, and is the leading cause of failures in drug development, boxed warnings on marketed drugs, and market withdrawals of approved drugs. Inhibition of BSEP and MDR3 is one of the underlying mechanisms for DILI. It has been an important task to develop physiologically relevant, reliable, higher throughput assays to determine the inhibition of BSEP and MDR3. Biotranex – Accurate. Efficient. Innovative Why BSEPcyte® • To address concerns about • BSEPcyte® is a physiologically other BSEP inhibition assays relevant, hepatocyte-based, – BSEP-transfected Sf9 insect cell high throughput assay membrane vesicles • Artificial membrane uptake system – Primary hepatocytes in • Incapable of evaluating BSEP suspension inhibition by metabolites • Physiologically relevant • Questionable physiologically – IVIVC relevance • Efflux – Human sandwich-cultured • hepatocytes In-situ metabolism • Reproducible, sensitive, dynamic • Technical challenges range • Low throughput • Flexible – Others • High-throughput • BSEP and sodium taurocholate transporter co-transfected kidney cells • Species comparison • Xenopus laevis oocytes • Canalicular membrane vesicles (CMV) Cheng Y, et al. Chem Biol Interact, 2015 Biotranex – Accurate. Efficient. Innovative BSEPcyte® Assay Principle Bile Acids (pKa: 5-6.5) Cholesterol CYP7A1 CYP27A1 CYP7B1 (Neutral BACS HSD3B7 Pathway) (Acidic Bile Acids VLCS CYP8B1 Pathway) AMACR AKR1D1 BCOX AKR1D4 BDP CYP27A1 Hepatocyte BAAT SCPx CA-CoA/CDCA-CoA Bile Salts BAAT BSEP Bile Salts (pKa: 1-4) >99% Bile salts exported by BSEP in human liver. CA: R1 = OH Jansen et al. Gastroenterology. 1999;117:1370-9 CDCA: R = H 1 BSEPcyte® Protected by US patent No 9,772,325 GCA/GCDCA: R2 = NHCH2COOH TCA/TCDCA: R = NHCH CH SO H 2 2 2 3 Information: [email protected] Physiological Bile Acid Conjugation Reactions DCA CA GCA GDCA TDCA TCA CDCA LCA GLCA GCDCA TCDCA TLCA Biotranex – Accurate. Efficient. Innovative BSEP IC50: BSEPcyte® vs Membrane Vesicles (MV) Total 86 drugs BSEPcyte® ➢ Detected more most-DILI- concern drugs than MV • metabolism-mediated • drug accumulation ➢ Produced physiologically relevant IC50 values ➢ Produced results highly correlated with cholestatic and mixed liver injury Zhang J, et al. Chem Biol Interact, 2016 Biotranex – Accurate. Efficient. Innovative BSEPcyte®: Most-DILI Concern Drugs Compound Name DILIConcern Label Section IC50 (µM) Cmax/IC50 ritonavir Most-DILI-Concern Warnings and precautions 0.2 37.8 deferasirox Most-DILI-Concern Box warning 11.9 14.2 troglitazone Most-DILI-Concern Withdrawn 0.5 12.8 cyclosporine Most-DILI-Concern Warnings and precautions 0.1 7.8 indinavir Most-DILI-Concern Warnings and precautions 2.8 4.7 ketoconazole Most-DILI-Concern Box warning 1.5 4.7 fasiglifam Most-DILI-Concern Discontinued 2.8 3.6 benziodarone Most-DILI-Concern Withdrawn 0.8 2.4 benzbromarone Most-DILI-Concern Withdrawn 2.8 1.5 etodolac Most-DILI-Concern Warnings and precautions 83.9 0.82 bosentan Most-DILI-Concern Box warning 9.2 0.81 lumiracoxib Most-DILI-Concern Withdrawn 45.6 0.69 leflunomide Most-DILI-Concern Box warning 50.0 0.50 disulfiram Most-DILI-Concern Warnings and precautions 12.0 0.45 diclofenac Most-DILI-Concern Warnings and precautions 31.8 0.21 bromfenac Most-DILI-Concern Withdrawn 93.3 0.19 itraconazole Most-DILI-Concern Warnings and precautions 3.1 0.13 ticlopidine Most-DILI-Concern Warnings and precautions 82.0 0.10 ciglitazone Most-DILI-Concern Discontinued 0.8 ? fipexide Most-DILI-Concern Withdrawn 4.9 (600 mg dose) saquinavir Less-DILI-Concern Warnings and precautions 0.4 13.6 rosiglitazone Less-DILI-Concern Warnings and precautions 0.2 5.2 pioglitazone Less-DILI-Concern Warnings and precautions 1.4 2.1 chlorpromazine Less-DILI-Concern Adverse reactions 6.4 0.15 trazodone Less-DILI-Concern Adverse reactions 43.0 0.12 BSEP IC50: BSEPcyte® vs Membrane Vesicles (MV) BSEPcyte ® MDR3cyte ® MV BSEP MRP2 MRP3 MRP4 Compound Name (uM) (uM) (uM) (uM) (uM) (uM) LOVASTATIN 2 17 19.3 133 133 133 REPAGLINIDE 1 18 22 72.3 16.7 53.9 FLUVASTATIN 1 >100 36.1 stimulation 57 133 NIFEDIPINE 2 23 64 133 133 16.6 DONEPEZIL 9 3 78 133 133 72.9 stimulation ACITRETIN 0.04 >100 38.2 133 49.00 /inhibition GLIMEPIRIDE >100 >100 15.7 stimulation 6.39 64 DANAZOL >100 >100 18.7 133 133 133 INDOMETHACIN 11 12 42 stimulation 66 5.8 Additional 250 drugs AMIODARONE >100 >100 43 133 133 133 ENTACAPONE 10 >100 55.6 133 35.1 6.8 BENZBROMARONE 0 1 17.5 41.6 133 17 INDINAVIR >100 >100 21.2 133 83 133 KETOCONAZOLE 0 1 3.4 133 133 69 CLOFIBRATE >100 >100 71 133 133 133 LOPINAVIR 0 >100 17.3 133 21 47 BSEPcyte® DICLOXACILLIN 7 >100 56.4 133 133 133 FENOFIBRATE >100 >100 15.3 133 133 133 ➢ Detected more most-DILI- PIOGLITAZONE <0.3 >100 0.4 stimulation 133 49.5 CHLORDIAZEPOXIDE 58 8 44.1 133 133 133 MELOXICAM 55 15 77.5 stimulation 133 133 QUINAPRIL >100 >100 94 stimulation 133 133 concern drugs than MV OXAPROZIN >100 >100 133 133 133 12 NITROFURANTOIN >100 62 133 133 133 20.9 NALIDIXIC ACID >100 >100 133 133 133 57.5 ACETAZOLAMIDE >100 >100 133 133 133 133 • metabolism-mediated ALLOPURINOL >100 >100 133 133 133 133 AMITRIPTYLINE 10 6 133 133 133 133 AMLODIPINE 1 4 133 133 133 133 AMOXICILLIN >100 >100 133 133 133 133 • drug accumulation AZATHIOPRINE >100 >100 133 133 133 133 CAPTOPRIL >100 >100 133 133 133 133 CARBAMAZEPINE 116 85 133 133 133 133 CARVEDILOL 1 0 133 133 133 133 ➢ Produced physiologically CEFACLOR >100 >100 133 133 133 133 CELECOXIB 12 >100 133 133 133 133 CHLORAMBUCIL 23 >100 133 133 133 133 CHLORAMPHENICOL >100 >100 133 stimulation 133 133 relevant IC50 values CHLORPHENIRAMINE 110 4 133 133 133 133 CHLORPROMAZINE 3 1 133 133 133 133 CHLORPROPAMIDE >100 >100 133 133 133 133 CIMETIDINE >100 >100 133 133 133 133 ➢ Produced results highly CIPROFLOXACIN >100 >100 133 133 133 133 CISPLATIN >100 1 133 133 133 133 CITALOPRAM 56 6 133 133 133 133 CLOZAPINE 10 1 133 133 133 133 correlated with cholestatic DESIPRAMINE 7 3 133 133 133 133 DOXEPIN 10 6 133 133 133 133 EFAVIRENZ 2 5 133 133 133 133 EPROSARTAN >100 >100 133 57.9 6.65 133 and mixed liver injury ERYTHROMYCIN 117 >100 133 133 133 133 ETHACRYNIC ACID 29 >100 133 3.85 95.9 133 FLUCONAZOLE >100 >100 133 133 133 133 FLUOXETINE 1 3 133 133 133 133 FLUTAMIDE 1 >100 133 133 133 133 GEMFIBROZIL 39 >100 133 stimulation 133 133 GLICLAZIDE >100 >100 133 133 133 133 GLIPIZIDE >100 >100 133 133 133 133 HALOPERIDOL >100 16 133 133 133 133 IMIPRAMINE 15 6 133 133 133 133 INDAPAMIDE 25 35 133 133 133 133 IPRONIAZID >100 >100 133 133 133 133 LAMIVUDINE >100 >100 133 133 133 133 LANSOPRAZOLE 8 12 133 133 133 133 LEFLUNOMIDE 20 88 133 133 133 133 LEVOFLOXACIN >100 >100 133 133 133 133 LOMUSTINE 21 30 133 133 133 133 MEROPENEM >100 >100 133 133 133 133 METHIMAZOLE >100 >100 133 133 133 133 METRONIDAZOLE >100 >100 133 133 133 133 MEXILETINE 268 122 133 133 133 133 MIRTAZAPINE 20 17 133 133 133 133 NAPROXEN >100 >100 133 133 133 133 NEVIRAPINE 73 >100 133 133 133 133 NIMESULIDE 7 16 133 133 133 133 NORTRIPTYLINE 15 1 133 133 133 133 OFLOXACIN >100 >100 133 133 133 133 OLANZAPINE >100 >100 133 133 133 133 OXCARBAZEPINE >100 >100 133 133 133 133 Yucha, et al. Toxicol Sci, 2017 PIROXICAM 17 >100 133 133 133 133 PREGABALIN >100 >100 133 133 133 133 PROPAFENONE 6 2 133 133 133 133 RAMIPRIL >100 >100 133 133 133 133 He, et al. (To be published) RANITIDINE >100 177 133 133 133 133 LAMOTRIGINE 126 74 133 not tested not tested not tested Biotranex – Accurate. Efficient. Innovative BSEPcyte®: Species Difference in BSEP Inhibition (IC50, µM) Drug Human Monkey Dog Rat Mouse Haloperidol 25.6 64 73 54 >100 Ticlopidine 82 >100 >100 >100 >100 Saquinavir 0.4 3.1 3.5 5.1 >100 Atorvastatin 2.6 0.6 0.9 0.2 31.7 Azathioprine >1000 >100 >100 >100 >100 Cerivastatin 3.9 0.99 8.8 1.9 26.5 Chlorpromazine 44.3 7.28 29.4 13.1 136.6 Cyclosporine 0.1 0.71 2.4 0.5 4.69 Enalapril >1000 >100 >100 >100 >100 Etodolac 83.9 >100 >100 >100 >100 Fenofibrate 61 126.5 89 178.6 >100 Glimepiride 14.1 5.3 12.3 1.5 >100 Griseofulvin 756.9 43.5 85.8 45.7 >100 Imipramine 185.2 106.1 171.3 169.3 >100 Ketoconazole 1.5 4.4 7.5 4.1 >100 Lovastatin 1.7 14 15.9 14.2 >100 Methimazole >1000 >100 >100 >100 >100 Norfloxacin >1000 >100 >100 >100 >100 Piroxicam 151.4 182.4 >100 >100 >100 Ritonavir 0.21 1.2 1.7 0.5 5.7 Rosiglitazone 0.23 5 24.4 9.5 >100 Sulindac 122.9 >100 >100 >100 >100 Trazodone 42.9 134 >100 186 >100 Troglitazone 0.48 1.4 4.6 4.9 65.4 Zhang J, et al.