The Center for Alternatives to Animal Testing is an academic center affiliated with the Division of Toxicological Sciences in the Department of Environmental Health Sciences of the Johns Hopkins University Bloomberg School of Public Health.
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TestSmart Pharmaceuticals
May 7-9, 2001
PIER 5 HOTEL
711 Eastern Avenue
Baltimore, Maryland
Sponsors: 3M, Bristol-Meyers Squibb Company, Huntingdon Life Sciences, Schering-Plough Research Institute, and Taconic Farms, Inc.
Abstract for TestSmart--Pharmaceuticals: An Efficient and Humane Approach to Predictors of Potential Toxic Effects of Drugs
Application Of Human Cells In The Evaluation Of Drug Toxicity
Albert Li
In Vitro Technologies, Inc.
Jerry Collins
FDA
Joseph Tomaszewski
NIH
Session 5: Human cells and drug toxicity evaluation
- Jerry Collins: Overview
- Joe Tomaszewski: Bone marrow cell toxicity
- Al Li: Human hepatotoxicity
- Panel discussion
Critical Determinants for Clinical Success of a Drug Candidate
- Pharmacological properties
- ADME-Tox Properties
- bioavailability
- metabolic stability
- toxicity
- drug-drug interaction potential
Optimization of Human Drug Properties
- Identify NCEs with pharmacological activities
- Screen with human-based in vitro assays for bioavailability, metabolic stability, toxicity, and drug-drug interaction potential
- Build a portfolio of NCEs with high probability of success in the clinic
Why Human In Vitro Systems?
- Human: Human-specific results that may not be obtained with nonhuman systems (e.g. laboratory rats)
- In Vitro: Testing can be performed with limited amounts (ug-mg) of test substance
Parameters Critical to Successful Testing with In Vitro Human Assays
- Apply experimental systems with human-specific properties relevant to the endpoints to be evaluated
- Perform experiments under physiologically relevant conditions
Human In Vitro ADME-Tox Assays
- Intestinal Absorption (Caco-2)
- Metabolic Stability (Human hepatocytes)
- Human metabolite profile (Hepatocytes)
- Toxicity (Hepatocytes; tissue slices)
- Drug-drug interaction potential (Human hepatocytes; liver microsomes)
Intestinal Absorption
- 3-Day Caco-2 absorption model developed
- Little difference between 3-day and the routine 21-day culture, including Pgp expression
- Robust assay with acceptable day-to-day variations
- Good correlation between in vivo absorption and 3-day Caco-2 results
3-Day Caco-2 assay can be used routinely for the screening of intestinal absorption
Roles of the Liver in Drug Properties
- Key organ for xenobiotic metabolism, therefore plays a critical role in metabolic stability, toxicological properties, and drug-drug interaction potential
- Often a target of drug toxicity
Drug-drug interactions and hepatotoxicity are two major reasons that drugs are taken off the market
In Vitro Models of the Liver
- Hepatocytes
- Liver slices
- Liver microsomes
- cDNA-microsomes
USFDA Guidance for Industry
"The most complete picture for hepatic metabolism can be obtained with liver systems, in which the cofactors are self-sufficient and the natural orientation for linked enzymes is preserved.
Isolated hepatocytes and precision-cut slices have these desirable features."
Guidance for Industry, Drug Metabolism/Drug Interaction Studies in the Drug Development Process: Studies In Vitro CDER, CBER, U.S. FDA, 1997
Why Human Hepatocytes?
- Known species-species differences in drug metabolism and toxicity
- Technology advances allowing isolation, culturing, and cryopreservation of highly viable and functional human hepatocytes
Cryopreservation of Human Hepatocytes*
- High viability and yield
- Similar xenobiotic metabolism as freshly isolated cells
- Responsive to CYP1A and CYP3A inducers
*Loretz, Li, Wilson (1989). Xenobiotica 19:489-498
*Li et al (1999), Chemico-Biol Interact 121:17-35
*Ruegg et al. (1997). In Vitro Toxicol. 10:217-222
Human Hepatocytes and Drug Metabolism
- Prediction of human metabolite profile
- Interspecies comparison in metabolism
- Estimation of human intrinsic clearance
- High-throughput screening (HTS) for metabolic stability
Human Hepatocytes and Toxicology
- Species-comparison in hepatotoxicity
- Dose-response and mechanistic evaluation to aid safety assessment
- Early toxicity screening of drug candidates
In Vitro Human Hepatotoxicity
- Freshly isolated human hepatocytes
- 24 hr treatment
- Cryopreserved human hepatocytes
- 4 hr treatment
Cytotoxicity Endpoints
- ATP content
- GSH content
- MTT metabolism
- Neutral red uptake
- Alamar blue metabolism
Mechanism-Based Evaluation of Drug-Drug Interaction Potential
- CYP isoform identification (microsomes)
- CYP inhibitory potential (microsomes, hepatocytes)
- CYP induction potential (hepatocytes)
Drug-Drug Interactions: Scientific and Regulatory Perspectives (Li, A. P., Editor). Volume 43, Advances in Pharmacology Series Academic Press, San Diego, USA
Why Human Hepatocytes for CYP Inhibition
- Modeling of intracellular accumulation: Cin>>Cout
- Modeling of intracellular removal (e.g. pGP): Cout>>Cin
- Physiological enzyme and cofactor concentrations
- Complete, undisrupted, multiple drug-metabolizing pathways
Mechanism-Based Evaluation of Drug-Drug Interaction Potential
- CYP isoform identification (microsomes)
- CYP inhibitory potential (microsomes, hepatocytes)
- CYP induction potential (hepatocytes)
Drug-Drug Interactions: Scientific and Regulatory Perspectives (Li, A. P., Editor). Volume 43, Advances in Pharmacology Series Academic Press, San Diego, USA
All Known Inducers in Human In Vivo Are Inducers in Human Hepatocytes In Vitro
- Carbamazepine
- Dexamethasone
- Omeprazole
- Phenobarbital
- Phenytoin
- Rifampin
- Troglitazone
IDT-Related Drugs:.CYP Induction Potential*
- Halothane
- Troglitazone(CYP3A)*
- Tacrine
- Procainamide(3A4)*
- Carbamazepine(3A4)*
- Phenytoin(3A4)*
- Hydralazine
- Amineptine
- Amodiaquine
- Felbamate(3A4)*
- Tienilic acid
- Rifampin(3A4)*
- Isoniazid(2E1)*
- Ethinyl Estradiol
Human Hepatocytes and Drug Development
- Procedures developed for isolation, culturing, and cryopreservation
- Phase I and II drug metabolism activities retained in cryopreserved human hepatocytes
- Assays developed for the application of human hepatocyes in metabolite profiling, metabolic stability, hepatoxicity, and drug-drug interactions
Human In Vitro Experimental Systems.
- 3-Day Caco-2 system for the modeling of intestinal uptake
- Cryopreserved and freshly isolated human hepatocytes for:
- metabolite profiling
- metabolic stability
- hepatotoxicity
- drug-drug interactions
Human Experimental Systems and Drug Development: Future Directions
- Continue to add new donors to the cryopreserved human hepatocyte bank
- Apply HTS assays with cryopreserved human hepatocytes with large variety of chemical structures
- Develop and validate approaches for accurate in vitro:in vivo extrapolation
- Continue to develop physiologically relevant experimental models for the evaluation of human drug properties: expression genomics, proteomics
Reduction, Refinement and Replacement of Animal Testing by Human In Vitro Systems
- Reduction: Minimize the usage of irrelevant animal species
- Refinement: Aid optimization of in vivo experimental design
- Replacement: Replacement of animal tests with in vitro human assays