Novel Cytochrome P450 Reaction Phenotyping for Low-Clearance Compounds Using the Hepatocyte Relay Method. Li Di, Ph.D. Pfizer Inc.

Transcription

1 Novel Cytochrome P450 Reaction Phenotyping for Low-Clearance Compounds Using the Hepatocyte Relay Method Li Di, Ph.D. Pfizer Inc. June 21, 2017

2 Challenges of Low Clearance Low clearance goal of drug discovery projects Enhance exposure Reduce dose Prolong half-life Low clearance nightmare of DMPK Under-prediction of half-life (e.g., 0.5 year, never wash-off) High projected dose due to over-prediction of CL Challenging phenotyping low confidence DDI prediction Unknown metabolites potential MIST issues..

3 Low Clearance Major Scientific Gap Increasing of low CL compounds HT-ADME and Met ID enable rapid SAR Effective Med Chem design strategy New chemical space: liver targeting New dosing paradigm: QW Internal Survey at Pfizer ~30% compounds are low CL in drug discovery portfolios For some projects, most compounds are low CL Lack of low CL tools in drug discovery Major scientific gap

4 Low Clearance Tools Pfizer Investigated 1.5 Decades! SCHH HHEP Relay.. rhcyp Liver Chip Hepregen HLM Relay Hurel. HLM Stablizers Induction

5 Novel Hepatocyte Relay Method Transfer Supernatant DMD, 2012, 57,

6 HHEP Relay IVIVE of Commercial Drugs Compounds Metabolizing Enzymes Human In Vivo CL int (ml/min/kg) Relay Method CL int (ml/min/kg) Fold Difference between In Vitro and In Vivo Diazepam CYP3A, 2C ± Disopyramide CYP3A ± Theophylline CYP1A ± Timolol CYP2D ± Tolbutamide CYP2C ± S-Warfarin CYP3A, 2C ± Alprazolam CYP3A ± Ranitidine CYP2D6, 1A2, 3A Zolmitriptan CYP1A2, MAO ± 1.9 * 3.3 * *Underprediction of in vivo clearance due to extrahepatic contribution of MAO

7 HHEP Relay IVIVE of Pfizer Compounds Therapeutic Area Compounds Human CL int, u HHEP Relay CL int, u ml/min/kg ml/min/kg Fold Difference CVMED A CVMED B Oncology C Oncology D Oncology E Oncology F * 3.5* (AO) Imflammation G 4.2 (CL H ) 3.2 (CL H ) 1.3 * Underprediction of in vivo clearance due to extrahepatic contribution of AO Prospective Prediction Oncology: 14.6 L/h (predicted), 11.7 (actual) Neuroscience: 0.9 ml/min/kg (predicted), 0.9 (actual) CVMED: 3.87 ml/min/kg (predicted), 3.95 (actual)

8 Importance of Reaction Phenotyping and CL Pathways Assess victim DDI Set inclusion / exclusion criteria Understand PGx, individual variability, co-med Impact confidence in CL prediction (non-cyp), IVIVE, dose, t 1/2 If incorrect. Safety concerns, irrelevant DDI studies Require much more work to figure out why Experimentally and modeling and simulation

9 Current Tools for Reaction Phenotyping of Low CL Compounds rcyp low power, problematic ISEFs, substrate dependent, non-physiological system specifically addresses CYP reaction phenotyping often only picks up CYP3A for low CL compounds misleading Metabolite standards chemical- or bio-synthesized, best applied with 1-3 metabolites/pathways major metabolites might not have high enough coverage for overall CL needs lead time and more suited for late stage development compounds Radio-labeled material need material, expensive, tedious & long process ~ 4 weeks has fallen out of favor with project teams HHEP relay with chemical inhibitors following substrate depletion No need to have metabolite standards or radio-labeled material No assumptions of clearance pathways

10 Ln % Remaining Ln % Remaining Challenges of Reaction Phenotyping for Low CL Compounds High CL + inh Low CL + inh > 90% - inh > 30% - inh Time Steep decline Large difference +/- inhibitor Can detect high f m (>0.9) Time Shallow decline Small difference +/- inhibitor f m limit is low (e.g. > 0.3)

11 Challenges of Reaction Phenotyping for Low CL In order to achieve 90% inhibition, the limit of CL int needs to be 10-fold lower than regular CL int measurement Assays CL int Limit (ml/min/kg) 90% Inhibition CL int (ml/min/kg) HLM (0.75 mg/ml, 1 hr) 7 70 HHEP Relay (0.5 Mcells/mL, 20 h) HHEP Relay (2 Mcells/mL, 20 h) Moderate-high CL might need relay assay to achieve 90% inhibition The CL int limit can be lower if multiple pathways are involved

12 Novel HHEP Relay Reaction Phenotyping Assay Add Substrate Transfer Supernatant Pre-incubation with Inhibitor Remove Inhibitor Incubate with Substrate Next Relay MBIs to avoid the impact of inhibitor depletion during long incubation MBIs were removed from incubation after inactivation to increase selectivity by minimizing reversible inhibition (CYP, AO, CES, UGT, SULT, Transporters) DMD, 44(3): 460-5, 2016

13 Experiment Conditions for HHEP Relay Phenotyping CYP Pre-Incub. Time (min) Inactivators and Concentrations Substrates Metabolites 1A µm Furafylline Phenacetin Acetaminophen 2B µm Phencyclidine Bupropion OH-Bupropion 2D µm Paroxetine Dextromethorphan Dextrorphan 2C8 30 µm Gemfibrozil Glucuronide Amodiaquine N-Desethylamodiaquine 2C µm Tienilic Acid Diclofenac 4 -OH-Diclofenac 2C µm Esomeprazole Mephenytoin 4 -OH-Mephenytoin 3A µm Troleandomycin Midazolam 1 -OH-Midazolam 3A µm CYP3cide Midazolam 1 -OH-Midazolam Pan-CYP 30 1 mm ABT & 15 µm Tienilic Acid All Above All Above DMD, 44(3): 460-5, 2016

14 % Inhibtion % Inhibition % Inhibition % Inhibition % Inhibition % Inhibition % Inhibition % Inhibition % Inhibition CYP Inactivactor Selectivity Furafylline (1A2) Phencyclidine (2B6) Paroxetine (2D6) A2 2B6 2C8 2C9 2C19 2D6 3A 0 1A2 2B6 2C8 2C9 2C19 2D6 3A 0 1A2 2B6 2C8 2C9 2C19 2D6 3A Gemfibrozil Glucuronide (2C8) Tienilic Acid (2C9) Esomeprazole (2C19) A2 2B6 2C8 2C9 2C19 2D6 3A 0 1A2 2B6 2C8 2C9 2C19 2D6 3A 0 1A2 2B6 2C8 2C9 2C19 2D6 3A Troleandomycin (3A) CYP3cide (3A4) 1-Aminobenzotriazole (Pan-CYP) A2 2B6 2C8 2C9 2C19 2D6 3A 2C9 0 1A2 2B6 2C8 2C9 2C19 2D6 3A 0 1A2 2B6 2C8 2C9 2C19 2D6 3A

15 Hepatocyte Relay Reaction Phenotyping Method Estimate contribution for 8 CYPs (1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A, 3A4) and pan-cyp 48 well plate format (can be higher density if needed), 150 L Cryopreserved human hepatocytes, 0.5 or 2 million cells/ml, suspension, multiple donors All inhibitors are MBIs (mechanism-based inactivator) After pre-incubation, remove the inhibitors to increase selectivity 1 µm substrate concentration, monitor substrate depletion Incubation time: 4 h/relay with 5 or less relays Report F m

16 HHEP Relay Reaction Phenotyping Assay Validation % Remaining Erythromycin + TAO 0 10 Reported: mostly 3A Relay f m: A Time (hour) Reported f m : A2 Relay f m: A2

17 % Remaining % Remaining HHEP Relay Reaction Phenotyping Assay Validation % Remaining Timolol (2D6) Reported: 2D6 major 2C9 minor Relay f m: D Time (hour) 0 Tolbutamide (2C9) 0 Tolbutamide + Tienilic Acid Reported f m : C Relay f m: C Time (hour) Time (hour)

18 HHEP Relay Reaction Phenotyping Assay Validation % Remaining % Remaining % Remaining Diazepam (3A + 2C19) Diazepam + TAO Time (hour) Time (hour) Diazepam + Esomeprazole 0 Reported f m : 0.5 3A, 0.5 2C Time (hour) Relay f m: A, C19 DMD, 44(3): 460-5, 2016

19 Relay Reaction Phenotyping Application Compound is low CL CL int = 11.8 ml/min/kg in HHEP relay Reaction phenotyping using MBI in the HHEP relay CYP3A f m = 0.34, total CYP 0.71, non-cyp 0.29 (SULT, UGT) Guide design for Clinical Development Plan Human itraconazole DDI study: - 87% increased in AUC - Estimated human CYP3A f m = 0.47 HHEP relay phenotyping data is consistent with clinical DDI results

20 Relay Reaction Phenotyping Application Compound is low CL CL int = 8.6 ml/min/kg in HHEP relay rcyp indicated high CYP3A F m > 0.99 Clinical DDI study with ketoconazole CYP3A F CL = 0.6 HHEP relay reaction phenotyping CYP3A F m = 0.76 (F CL = 0.68, 10% renal CL) CYP2C8 F m = 0.15 Total CYP > 0.91 (minor UGT pathway in humans ~5%) HHEP relay phenotyping data is consistent with clinical DDI results

21 Reaction Phenotyping with HHEP Relay Assay Drug candidate is low CL CL int = 2.8 ml/min/kg in HHEP relay Assume all CYP3A based on rcyp data and 1 st compound in the clinic HHEP relay reaction phenotyping CYP3A F m = 0.36 Non-CYP ~ 0.60 Follow up studies suggested AO involvement. Data is consistent with azaheterocyclic structure. HHEP relay reaction phenotyping is critical in diagnosing non-cyp pathway in order to guide clinical DDI studies and to predict clearance and dose

22 Reaction Phenotyping with HHEP Relay Assay Drug candidate is low clearance CL int = 6.5 ml/min/kg in HHEP relay Assume mostly CYP pathways based on metabolite ID data HHEP relay reaction phenotyping CYP3A F m = 0.21 Non-CYP F m = 0.73 Follow up studies suggested involvement of reductases in the clearance pathway MIST coverage, clearance prediction, individual variability, PGx, etc. HHEP relay reaction phenotyping is powerful in diagnosing non-cyp pathways

23 HHEP Relay Reaction Phenotyping Non-CYP clearance pathways continue to increase due to effective HT-HLM screening and early met ID HHEP is physiologically relevant system with a complete complement of CYP and non-cyp enzymes and all the cofactors HHEP reaction phenotyping with or without the relay format is highly effective in determining competing clearance pathways HHEP relay reaction phenotyping assay Flexible - One can change cell densities and number of relays depending on compound properties Does not require metabolite standards or radio-labeled material or clear understanding of clearance pathways Can be implemented early in drug discovery

24 CYP Reaction Phenotyping Screening Strategies Verification rcyp Metabolite Profile Metabolite Formation Docking Structure Information HHEP HHEP Relay Radio-Labeled High / Moderate CL Moderate / Low CL Very Low CL

25 Shift of Property Space: Consequence of HT-HLM Screening? CYP Non-CYP High CL CYP High CL Non-CYP CL HHEP Relay Low CL CYP Low CL Non-CYP Challenging Space (Knowledge Gap) IVIVE, PGx, Tissue distribution Species difference DDI, inducibility, Individual variability Disease state

26 Summary HHEP Relay Assay Good in vitro-in vivo correlation in all species/strains Use cryopreserved hepatocytes of multiple donors, genotyped HHEP Suspension format, no cell culture is needed 4 hour incubation no need to be on consecutive days Can be done anytime no lag time waiting for culture plates Flexible different cell density, species, donor, incubation time Low cost extension of the regular hepatocyte assay Applicable to CL int, met ID and reaction phenotyping Can be readily setup in most DMPK labs

27 References- HHEP Relay Li Di, Patrick Trapa, R. Scott Obach, Karen Atkinson, Yi-An Bi, Angela C. Wolford, Beijing Tan, Thomas S. McDonald, Yurong Lai, Larry M. Tremaine. A Novel Relay Method for Determining Low-Clearance Values. DMD, 2012, 40(9): Li Di, Karen Atkinson, Christine C. Orozco, Carrie Funk, Hui Zhang, Thomas S. McDonald, Beijing Tan, Jian Lin, Cheng Chang, R. Scott Obach, In Vitro in Vivo Correlation for Low Clearance Compounds Using Hepatocyte Relay Method, DMD, 2013, 41, Eric Ballard, Christine Orozco, Scott Obach, Generation of Major Human Excretory and Circulating Drug Metabolites Using a Hepatocyte Relay Method, DMD, 2014, 42(5): Li Di, Scott Obach, Addressing the Challenges of Low Clearance in Drug Research, AAPS J, 17(2):352-7, 2015 Xin Yang, Karen Atkinson, Li Di, Novel Cytochrome P450 Reaction Phenotyping for Low Clearance Compounds Using Hepatocyte Relay Method, DMD, 2016, 44(3):

28 Acknowledgements Xin Yang Karen Atkinson Christine Orozco Eric Ballard Patrick Trapa Larry Tremaine Tess Wilson HDO, BioTrans, PRs and Management