In Vitro ADMET Laboratories Inc. A Hepatocyte Enterocyte Research Organization. Columbia, MD and Malden, MA

Transcription

1 In Vitro ADMET Laboratories Inc. A Hepatocyte Enterocyte Research Organization Columbia, MD and Malden, MA

2 In Vitro ADMET Laboratories (IVAL) Locations: Columbia, MD and Malden, MA Date of Incorporation: November, 2004 Mission: To provide products and contract research service to enhance the efficiency of drug development 2

3 IVAL Activity Develop and provide physiologically relevant in vitro experimental systems Organ-specific properties Species-specific properties Develop experimental approaches to aid accurate assessment of human drug properties 3

4 IVAL Scientific Team David Ho (previously Takeda, Cambridge) Qian Yang (previously NCI) Ivy Wei (previously NIH) Walter Mitchell (previously Pfizer, Groton) Carol Loretz (previously University of Washington, Seattle) Kirsten Amaral (previously Life Technologies, RTP) Albert P. Li (previously Monsanto/G. D. Searle) 4

5 Albert P. Li, Ph. D. Pioneered cryopreservation of human hepatocytes Pioneered cryopreservation of human enterocytes and intestinal mucosa 5

6 IVAL Products In Vitro Liver and Intestine Models Liver Fully functional plateable cryopreserved human hepatocytes (999Elite ) MetMax cryopreserved hepatocytes Intestine Cryopreserved enterocytes MetMax cryopreserved enterocytes Cryopreserved intestinal mucosa 6

7 IVAL Plateable Cryopreserved Hepatocytes Human Cynomolgus Monkey Beagle Dog CD-1 Mouse SD Rat Wistar Rat 7

8 999-Elite Cryopreserved Human Hepatocytes >90% viability >90% confluency >9 days culture duration

9 Applications of 999-Elite Cryopreserved Human Hepatocytes in Drug Development DMPK Metabolism Uptake Efflux Drug-Drug Interactions (Induction) Toxicology Hepatotoxicity screening Elimination of sdili potential Species selection for safety studies Pharmacology Hepatitis B NASH Cholesterol synthesis

10 Pooled donor human hepatocytes 1. Pooling of hepatocytes from multiple donors allow the generation of experimental results representative of a generalized population 2. Preparation of Pooled Donor Human Hepatocytes involves thawing of cryopreserved human hepatocytes from individual donors, followed by pooling and re-cryopreservation 3. Use of pooled donor human hepatocytes is a common practice for drug metabolism studies

11 QuickFreeze Pooled Human Hepatocytes 1.High viability 2.Plateable (if pooling plateable lots) Patent allowed: United States, China Patent pending: Europe

12 MetMax Human Hepatocytes Permeabilized, cofactor supplemented hepatocytes An experimental system with the advantages of hepatocytes and the ease of operation and robustness of cell free systems 12

13 Activity (pmol/min/million hepatocytes) Comparison of 16 Drug Metabolizing Enzyme-Selective Substrates Intact (PHH; blue) Vs MetMax (PHHX; red) Hepatocytes: PHH PHHX Drug Metabolizing Enzyme Pathway 13

14 Isolation and cryopreservation of enterocytes from human small intestines Enterocytes Mucosal Epithelium 14

15 DME Activities of Human Enterocytes Lot Number Gender Ethnicity Age CYP2C9 CYP2C19 CYP3A4 UGT SULT 2J2 CES2 HE3005 Male C HE3006 Female C HE3007 Male H HE3008 Male C HE3009 Female C HE3010 Male C HE3011 Female C HE3013 Female AA 57 NA NA 0.2 NA NA NA NA HE3014 Male AA HE3015 Male C HE3016 Male AA HE3019 Male C HE3020 Male C HE3021 Male AA HE3027 Female C HE3028 Male AA HE3029 Male C HE3031 Female C

16 Drug Metabolism Activities (pmol/min/mg protein) of CHIM CHIM6001 (Duodenum) CHIM6003 (Jejunum) CHIM6005 (Ileum) CYP1A CYP1A CYP2A CYP2B CYP2C CYP2C CYP2C CYP2D CYP2E CYP3A4-1 (Midazolam) CYP3A4-2 (Testosterone) ECOD UGT (7HC) SULT (7HC) GST (APAP) UGT (APAP) SULT (APAP) FMO MAO AO NAT NAT J CES

17 In Vitro ADME Uptake Metabolic stability DDI P450 Inhibition P450 Induction In Vitro Tox Hepatotoxicity Enterotoxicity Contract Service 17

18 Hepatocyte Uptake Assay Add Test Article Suspension Culture Attached Culture Add Test Article Incubate, 37 deg. C 1. Add to silicon oil tube 2. Centrifuge 3. Cut cell pellet 4. Extract cell pellet 5. LC/MS quantification 1. Remove medium 2. Wash 3. Extract cells 4. LC/MS quantification

19 Plated Human Hepatocytes Uptake Assay: Time-dependent uptake inhibited by rifampicin Collaboration with Yuichi Sugiyama (Riken) Substrate:PTV (0.1 μm), RSV(0.1 μm), CRV(0.1 μm), PRV(2 μm) Temperature:37 Time point: 0.25, 1.25, 2.5, 5, 15, 45, 90min Lot: HH1103 Control 100 μm Rifampicin

20 PREDICTION OF IN VIVO HEPATIC CLEARANCE

21 Hepatocyte Metabolic Stability Assay Add Test Article Suspension Culture: Pooled Donor Human Hepatocytes MetMax Pooled Donor Human Hepatocytes Attached Culture: Plateable Pooled Donor Human Hepatocytes Add Test Article Incubate, 37 deg. C 1. Add acetonitrile 2. LC/MS quantification of parent disappearance 3. Calculate in vitro intrinsic hepatic clearance (Clhep, vitro) 4. Estimate in vivo hepatic clearance (Clhep, vivo)

22 In Vitro Hepatic Clearance MetMax Pooled Donor Human Hepatocytes accurately predict human hepatic clearance in vivo (PHS9001: Pooled Human Hepatocytes; PHX 8001: MetMax Pooled Human Hepatocytes; Collaboration with Gary Hingorani and Karin Brown, Array Biopharma) In Vivo Systemic Clearance PHS900 1 y = x R² = 0.13 MetMa x PHHX 8001 y = x R² = Test article concentration: 1 um Hepatocyte: 1 million cells/ml Time points: 0, 5, 15, 30, 45 Endpoint: T1/2; Prediction: In vivo intrinsic hepatic clearance

23 U. S. Patent allowed in August, 2016 A Novel Plated Hepatocyte Relay Assay (PHRA) for In Vitro Evaluation of Hepatic Metabolic Clearance of Slowly Metabolized Compounds Drug Metabolism Letters, 2015, Volume 9 (E-pub ahead of print) Author(s): Chi-Chi Peng, Utkarsh Doshi, Chandra Prakash and Albert P Li. Affiliation: In Vitro ADMET Laboratories LLC, 9221 Rumsey Road, Suite 8 Columbia, MD 21045, USA Modification of Pfizer (Li Di s) Relay Assay 23

24 Removal of medium, pool for analysis and use for relay incubation Acetonitrile addition (estimate nonspecific binding) Pool Incubated medium Relay: Addition of incubated medium to a new hepatocyte plate Old plate LC/MS-MS New hepatocyte plate prepared 4 h before use 24

25 Plated Hepatocyte Relay Assay Accurately Predicts In Vivo Hepatic Clearance: A (within 2-fold); B (within 10-fold); D (>10 fold) Compound CL nonrenal, blood (ml/min/kg) Adjusted CL h, Fu=1 (ml/min/kg) Adjusted In vitro CL h, Fu=actual (ml/min/kg) Adjusted in vitro/in vivo ratio; Fu=1 Adjusted in vitro/in vivo ratio; Fu=actual Classification (Fu=1; adjusted) Classification (Fu=actual; adjusted) Ultra Low Clearance Compounds Tenoxicam D B Warfarin D A Meloxicam D B Tolbutamide A B Disopyramide A A Low Clearance Compounds Glimepiride (I) B A Glimepiride (II) B A Prednisolone B A Ibuprofen B A Riluzole A A Quinidine A A Clozapine A B Voriconazole A A Dexamethasone A A Prednisone A A Risperidone B A 25

26 P450 Induction 26

27 Human Hepatocyte P450 Induction (Gene Expression) CYP1A2 induction fold Treatment HH1121 HH1142 HH1144 Omeprazole (µm) Mean SD Mean SD Mean SD CYP2B6 induction fold Treatment HH1121 HH1142 HH1144 Phenobarbital (µm) Mean SD Mean SD Mean SD CYP3A4 induction fold Treatment HH1121 HH1142 HH1144 Rifampin (µm) Mean SD Mean SD Mean SD

28 Human Hepatocyte CYP2C Induction CYP2C8 induction fold Treatment HH1121 HH1142 HH1144 Rif (µm) Mean SD Mean SD Mean SD CYP2C9 induction fold Treatment HH1121 HH1142 HH1144 Rif (µm) Mean SD Mean SD Mean SD CYP2C19 induction fold Treatment HH1121 HH1142 HH1144 Rif (µm) Mean SD Mean SD Mean SD

29 Fold Induction Fold Induction Novel Technology: Human Enterocyte P450 Induction Vitamin D Induction of P450 Gene Expression in CHIM (Duodenum; 24 hr treatment) CYP3A4 Induction by 1,25(OH)2D CYP24A1 Induction by 1,25(OH)2D Concentration (nm)

30 In Vitro Toxicity Screening 30

31 Hepatocyte Hepatotoxicity Assay Attached Culture: Single Donor or Pooled Donor Human Hepatocytes Incubate, 37 deg. C 1. Culture for 4 hrs (preferred) to 24 hrs (maximum duration) to allow attachment 2. Add test article 3. Incubate with test article for 1 (routine) -7 days (medium changed daily) 4. Quantify cytotoxicity endpoints: i. Cellular ATP (cell viability; mitochondrial functions) ii. Reduced GSH (reactive metabolite formation) iii. Reactive oxygen species (ROS; oxidative stress) iv. Caspase 3/7 activation (apoptosis)

32 Percent Viability (% of control) Percent Viability (% of control) Multiple Species Hepatocyte Cytotoxicity Assay (ATP): Over-prediction (left) and Under-prediction (right) of Human Hepatotoxicity with Nonhuman Hepatocytes 150 Cyclophosphamide 150 Tacrine Concentration [ M] Concentration [ M] HUMAN CYNOMOLGUS MONKEY SD RAT CD-1 MOUSE BEAGLE DOG

33 Identification of sdili* Drugs *severe drug-induced liver injuries (liver failure)

34 Examples of Regulatory Actions on Marketed Drugs due to DILI( ) (Source: Mark Avigan, FDA, 201 Withdrawals (US* &/or other countries**) Boxed Warnings (US) troglitazone* lamividine bromfenac* leflunomide trovofloxacin* propylthiouracil tilbroquinol** pemolinet** tetrabamatet** tenofovir ebrotidinet** nefazodone** tolrestat** droxicam** sunitimib tolcapone bosentan deferasirox lapatanib pazopanib tipranavir

35 Se nsitiv ity % Evaluation of the Prediction Potential ROC of AUC ROS (sdili) ATP (sdili) GSH (sdili) Casp (sdili) ROS (odili) sdili ROS/AT P GSH ATP Casp3/7 Area P value < < Cut-off > < < > Sensitivity% % CI 74-98% 47-81% 64-93% 55-88% Specificity% % CI % 37-66% 60-86% 45-74% LR % - Specificity% odili ROS/AT P GSH ATP Casp3/7 Area P value < ROS: ROS/ATP ration Cut-off > < < > Sensitivity% % CI 65-89% 47-81% 61-86% 61-86% Specificity% % CI 51-87% 37-66% % 34-73% LR

36 In Vitro Enterotoxicity Screening Acetaminophen (APAP) and Naproxen Enterotoxicity in Cryopreserved Human Intestinal Mucosa from Three Donors: IC50 Values (mg/ml) Drug Duodenum (Donor 1) Jejunum (Donor 1) Duo+Juj+Ile (Donor 2) Duodenum (Donor 3) Jejunum (Donor 3) Ileum (Donor 3) APAP Naproxen

37 IVAL Activity Provision of physiologically relevant products Organ-specific properties Species-specific properties Provision of contract research service to aid accurate assessment of human drug properties 37

38 Contact Information Albert P. Li, Ph. D., President and CEO: George Amaral, U. S. Sales and Marketing: Bez Emadi, European Sales and Marketing: Deepak Barot, Indian Sales and Marketing: Nozomi Mizuno, Japanese Sales and Marketing: 38