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. Chem Biol Interact, 2016 Biotranex – Accurate. Efficient. Innovative BSEPcyte® Assay Platform Summary • BSEP function preserved in • BSEPcyte® primary hepatocyte – Physiologically relevant suspension system – Lot variations • In vitro-in vivo extrapolation – Timing – In situ metabolism capability – Components – Uptake transport and hepatocyte accumulation • Bile salts are exported – Pooled or individual donor primarily by BSEP in human hepatocytes hepatocytes – Applicable to all bile salts • BSEP inhibition – Cross species comparison – Highly associated with – Large dynamic range cholestatic and mixed DILI – Accurate, robust, – Species difference reproducible, and customizable – Higher throughput
BSEPcyte®, US patent 9,772,325
Information: [email protected] Why MDR3 ➢ Physiologically important: MDR3 is the primary mechanism for transporting phosphatidylcholine (PC) from hepatocyte to bile – PC plays an pivotal role in bile formation – PC forms mixed micelles with bile salts • Solubilize cholesterol • Reduce cytotoxicity of high concentration of bile salts – Maintain lipid asymmetry of hepatocyte membrane ➢ Validated target with clinical phenotype: MDR3 mutations lead to liver diseases – Progressive Familial Intrahepatic Cholestasis 3 (loss –of-function mutations) • Liver failure, death – Associated with intrahepatic cholestasis of pregnancy – Several mutations increase risk of liver diseases • MDR3 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 MDR3 is one of the underlying mechanisms for DILI.
Biotranex – Accurate. Efficient. Innovative MDR3 and Phosphatidylcholine
• MDR3 (ABCB4) R2 – ABCB4 gene – 1279 amino acid glycoprotein
– 86% similarity to ABCB1 (p-gp) R1 – Functional partner with ABCB11 (BSEP) – Mdr2 in mouse • Phosphatidylcholine of – Mainly in liver hepatocytes • Hepatocyte canalicular membrane – Primarily transported from – Transport phosphatidylcholine inner to outer by MDR3 from inner to outer leaflet of – ~98% of phospholipids in bile hepatocytes – Forms mixed micelles with bile • floppase salts and cholesterol • Prevents bile salt toxicity • Solubolizescholesterol – Maintain hepatocyte membrane lipid asymmetry
Biotranex – Accurate. Efficient. Innovative MDR3: Mechanism of Action
Biotranex – Accurate. Efficient. Innovative MDR3 Mutations an Liver Diseases: Large-scale whole-genome sequencing of the Icelandic population
We imputed these variants into 104,220 individuals down to a minor allele frequency of 0.1% and found a recessive frame shift mutation in MYL4 that causes early-onset atrial fibrillation, several mutations in ABCB4 that increase risk of liver diseases and an intronic variant in GNAS associating with increased thyroid- stimulating hormone levels when maternally inherited. Gudbjartsson et al, 2015 Biotranex – Accurate. Efficient. Innovative Why MDR3cyte®
• No other suitable MDR3 • MDR3cyte® is a inhibition assays physiologically relevant, available for drug hepatocyte-based assay screening – Primary hepatocytes in suspension – MDR3 transfected cells • Physiologically relevant • LLC-PK1, HEK293 – IVIVC • Efflux – MDR3-transfected Sf9 • In-situ metabolism insect cell membrane • Reproducible, sensitive, dynamic vesicles range • Flexible • No successful so far • High-throughput • Species comparison
Biotranex – Accurate. Efficient. Innovative MDR3cyte®: Working Principle
• Hepatocytes – PC: labeled by d9-choline – MDR3: activated by bile salts • d9-PC detected by LC- MS/MS
MDR3cyte® Protected by US patent No 10,280,401 Information: [email protected] MDR3cyte®: Phosphatidylcholine Species Transported from Human Hepatocytes to Extracellular Media -Similar Profile to Human Bile
XIC of +MRM (9 pairs): 768.0/193.0 amu from Sample 1 (HH_D9-choline_100 ug/mL ) of 150110_HH_MDR3_016.wiff (Turbo Spray), ... Max. 5.7e4 cps.
6.0e4 34:2
3.52 5.5e4
5.0e4
4.5e4
4.0e4
3.5e4
3.0e4 34:1 Intensity, cps Intensity,
2.5e4
2.0e4 36:4
1.5e4 38:6 36:2 1.0e4 36.3
5000.0 36:1
0.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5 4.6 Time, min Major PC in bile: 1-palmitoyl 2-linoleyl (C16:0/C18:2) and 1-palmitoyl 2-oleol (16:0/18:1)
Biotranex – Accurate. Efficient. Innovative He K, et al. Chem Res Tox, 2015 MDR3cyte®: MDR3 Inhibition by Drugs Associated with DILI Drug DILI Type BSEP (IC50, µM) MDR3 (IC50, µM) Human Human Monkey Rat Biotin Non-DILI >1000 >1000 >1000 >1000 Haloperidol CHL/VBDS 25.6 11 80 >100 Furosemide Non-DILI >1000 >1000 >1000 >1000 Ketoconazole Mixed 1.1 5.6 12 4.8 Saquinavir CHL 0.4 13 12 8.4 Chlorpromazine CHL/VBDS 44.3 9.7 6.0 2.7 Clotrimazole CHL 1.1 4.6 8.6 5.7 Cyclosporine CHL 0.1 >100* >100 >100 Imipramine Mixed 185 14 14.6 N/A Penicillamine Non-DILI >1000 >1000 >1000 >1000 Ritonavir CHL 0.2 9.6 N/A N/A Trazadone Mixed 43 >100 47 N/A Troglitazone CHL 0.5 10 6.7 N/A Verapamil Mixed 6.3 15 N/A
He K, et al. Chem Res Tox, 2015 Biotranex – Accurate. Efficient. Innovative MDR3cyte®: MDR3 Inhibition by 125 Drugs
B. ➢ MDR3 inhibition was determined using MDR3cyte® in human
hepatocytes
(µM) , total total ,
max ➢ MDR3 inhibition is C
associated with DILI Plasma Plasma ➢ Inhibition of both BSEP and MDR3 is associated with most severe DILI
MDR3 IC50 (µM)
Aleo, et al. Chem Res Toxicol. 2017 Biotranex – Accurate. Efficient. Innovative MDR3cyte®: Cmax/IC50 Ratio >0.2 Associated Severe DILI Drugs Drug_Name_LTKB DILI_Label_Sections MDR3 IC50 (μM) Cmax/IC50 Benzbromarone Withdrawn 0.4 9.86 Tipranavir Boxed Warning 15.6 6.07 Pazopanib Boxed Warning 18.0 3.81 Benzarone Withdrawn 4.0 3.76 Ketoconazole Boxed Warning 4.8 2.63 Benziodarone Withdrawn 2.3 2.35 Troglitazone Withdrawn 2.7 2.35 Ritonavir Warnings & Precautions 11.3 1.75 Benoxaprofen Withdrawn 112.7 1.39 Leflunomide Boxed Warning 22.6 1.10 Pirprofen Withdrawn 86.2 1.06 Nefazodone Boxed Warning 8.4 0.81 Lapatinib Boxed Warning 5.5 0.76 Zimeldine Withdrawn 1.2 0.52 Ebrotidine Withdrawn 3.8 0.48 Tasosartan Withdrawn 9.0 0.43 Tolcapone Boxed Warning 58.5 0.36 Methotrexate Boxed Warning 3.1 0.25
Biotranex – Accurate. Efficient. Innovative MDR3cyte®: Drugs with Higher Plasma Cmax/IC50 Ratio Associated with DILI
1.8 10 Most-DILI-concern Most-DILI-Concern 9 No-DILI-concern 1.6 No-DILI-Concern 8 1.4 Drugs with IC50 < 100 µM 7 1.2 6
1 /IC50 ratio /IC50 5
0.8 Cmax 4 ratio/I150 0.6 3 Cma Plasma 2 0.4 Mean ± SE 1 Plasma 0.2 0 0 0 50 100 IC50 (µM)
Biotranex – Accurate. Efficient. Innovative MDR3cyte® Assay Platform Summary • MDR3 function • MDR3cyte® preserved in primary – Physiologically relevant hepatocyte suspension holistic system – Lot variations • In vitro-in vivo extrapolation – Timing – In situ metabolism – Components capability • Phosphatidylcholines are – Uptake transport and transported primarily by hepatocyte accumulation MDR3 in human – Pooled or individual donor hepatocytes hepatocytes • MDR3 inhibition – Cross species comparison – Highly associated with DILI – Large dynamic range – Species difference – Accurate, robust, reproducible, and customizable MDR3cyte® Protected by US patent No 10,280,401 Information: [email protected] Acknowledgment • Biotranex LLC • Bristol-Myers Squibb – Thomas Woolf – Yaofeng Cheng – Lining Cai – Jinping Gan – Qin Shi – Williams Humphreys • NCTR/FDA • Takeda – Jie Zhang – Mingxiang Liao – Weida Tong – Cindy Xia • IVAL • Pfizer – Albert Li – A. David Rodrigues – Qian Yang – Michael Aleo
Contact: Kan He, [email protected] Backup Slides Expression Level of BSEP in Plasma Membrane of Human Hepatocytes in Suspension
Kumar V, et al. A Comparison of Total and Plasma Membrane Abundance of Transporters in Suspended, Plated, Sandwich-Cultured Human Hepatocytes Versus Human Liver Tissue Using Quantitative Targeted Proteomics and Cell Surface Biotinylation. DMD, 2019: 47: 350 Immunofluorecent Detection of Transporters in Cryopreserved Human Hepatocytes
“The large majority of transporters (note: including BSEP) seemed to be localized to the plasma membrane, whereas intracellular staining was comparatively weak. “
Lundquist P, EnglundG, Skogastierna C, LoofJ, Johansson J, Hoogstraate J, et al. Functional ATP-binding cassette drug efflux transporters in isolated human and rat hepatocytes significantly affect assessment of drug disposition. Drug Metabolism and Disposition 2014; 42:448-58. Bile Salt Synthesis and Biology • Multiple enzymes and steps – Hydroxylation by CYP7A1 is the rate limited step Cholesterol • Multiple subcellular organelles CYP7A1 CYP27A1 – ER, cytosol, mitochondria, peroxisomes CYP7B1 (Neutral BACS HSD3B7 • Majority of bile salts reabsorbed Pathway) (Acidic VLCS CYP8B1 Pathway) • Important biological process AMACR AKR1D1 – ~500 mg/day in human BCOX AKR1D4 • ~95% re-absorption BDP CYP27A1 – SCPx Osmotic bile flow – CA-CoA/CDCA-CoA Mixed micelles with phospholipids – Absorption of fat and vitamins BAAT • Bile salts for gene regulation – Ligands for FXR and TGR5 • Transporters involved in bile salt disposition – BSEP for bile salts transport to bile – NTCP for uptake transport – Others: ASBT, OST, MRP2/3/4, OATP1B1/2 • Other pathways CA: R = OH 1 – Secondary bile salts CDCA: R1 = H GCA/GCDCA: R2 = NHCH2COOH – Sulfate and glucuronide conjugates of BA TCA/TCDCA: R2 = NHCH2CH2SO3H Bile Salt Export Pump (BSEP) • Encoded by ABCB11 gene – 1321 AA – 12 transmembrane domains – Glycoprotein, 160 kDa • Expressed in canalicular plasma membrane of hepatocyte • Responsible for ATP-dependent bile salt transport – Predominant • <1% bile salts in bile of PFIC-2 patient vs normal • Like “vacuum cleaner” to keep low intracellular level of bile salts – Drive force for enterohepatic circulation of bile salts • Rate limited step • Substrate specificity for bile salts Kubitz, et al. 2012 – A few exceptions BSEPcyte®: BSEP Activity Variation in Human Hepatocytes
2.5
2.0
1.5
1.0
0.5 BSEP activity (nmole/million cells/h) (nmole/million BSEPactivity 0.0 Lot-Lot-Lot-Lot-Lot-Lot-Lot-Lot-Lot-Lot-Lot-Lot-Lot-Lot-Lot-Lot-Lot-Lot-Lot-Lot-Lot- 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20 21 Note: Both pooled and individual hepatocytes can be used with BSEPcyte® method Collaboration with Dr. Albert Li at IVAL Biotranex – Accurate. Efficient. Innovative