Evaluation of seven drug metabolisms and clearances by cryopreserved human primary hepatocytes cultivated in microfluidic biochips

We present characterization of the metabolic performance of human cryopreserved hepatocytes cultivated in a platform of parallelized microfluidic biochips. The RTqPCR analysis revealed that the mRNA levels of the cytochromes P450 (CYP 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4) were reduced after the adhesion period (when compared to the post-thawing step). The microfluidic perfusion played a part in stabilizing and partially recovering the levels of the HNF4α, PXR, OAPT2, CYP 1A2, 2B6, 2C19 and 3A4 mRNA on contrary to non-perfused cultures. Fluorescein diacetate staining and P-gp mRNA level illustrated the hepatocytes’ polarity in the biochips. Drug metabolism was assessed using midazolam, tolbutamide, caffeine, omeprazole, dextromethorphan, acetaminophen and repaglinide as probes. Metabolite detection and quantification revealed that CYP1A2 (via the detection of paraxanthine), CYP3A4 (via 1-OH-midazolam, and omeprazole sulfone detection), CYP2C8 (via hydroxyl-repaglinide detection), CYP2C19 (via hydroxy-omeprazole detection) and CYP2D6 (via dextrorphan detection) were functional in our microfluidic configurations. Furthermore, the RTqPCR analysis showed that the drugs acted as inductors leading to overexpression of mRNA levels when compared to post-thawing values (such as for HNF4α, PXR and CYP3A4 by dextromethorpahn and omeprazole). Finally, intrinsic in vitro biochip clearances were extracted using a PBPK model for predictions. The biochip predictions were compared to literature in vitro data and in vivo situations.

[1]  Cécile Legallais,et al.  A cocktail of metabolic probes demonstrates the relevance of primary human hepatocyte cultures in a microfluidic biochip for pharmaceutical drug screening. , 2011, International journal of pharmaceutics.

[2]  Laurent Griscom,et al.  Improvement of HepG2/C3a cell functions in a microfluidic biochip , 2011, Biotechnology and bioengineering.

[3]  L. Schwarz,et al.  Transcellular transport of fluorescein in hepatocyte monolayers: evidence for functional polarity of cells in culture. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[4]  Shufang Zhang,et al.  A robust high-throughput sandwich cell-based drug screening platform. , 2011, Biomaterials.

[5]  A. Guillouzo,et al.  Liver cell models in in vitro toxicology. , 1998, Environmental health perspectives.

[6]  M. Yarmush,et al.  Evaluation of a microfluidic based cell culture platform with primary human hepatocytes for the prediction of hepatic clearance in human. , 2009, Biochemical pharmacology.

[7]  Hugh A. Barton,et al.  Mechanistic Pharmacokinetic Modeling for the Prediction of Transporter-Mediated Disposition in Humans from Sandwich Culture Human Hepatocyte Data , 2012, Drug Metabolism and Disposition.

[8]  Hayley S. Brown,et al.  Primary Hepatocytes: Current Understanding of the Regulation of Metabolic Enzymes and Transporter Proteins, and Pharmaceutical Practice for the Use of Hepatocytes in Metabolism, Enzyme Induction, Transporter, Clearance, and Hepatotoxicity Studies , 2007, Drug metabolism reviews.

[9]  M. Toner,et al.  Effects of oxygenation and flow on the viability and function of rat hepatocytes cocultured in a microchannel flat-plate bioreactor. , 2001, Biotechnology and bioengineering.

[10]  John C Lipscomb,et al.  Scaling factors for the extrapolation of in vivo metabolic drug clearance from in vitro data: reaching a consensus on values of human microsomal protein and hepatocellularity per gram of liver. , 2007, Current drug metabolism.

[11]  M. Yarmush,et al.  A microfluidic hepatic coculture platform for cell-based drug metabolism studies. , 2010, Biochemical pharmacology.

[12]  Peter J H Webborn,et al.  Prediction of the Pharmacokinetics of Atorvastatin, Cerivastatin, and Indomethacin Using Kinetic Models Applied to Isolated Rat Hepatocytes , 2008, Drug Metabolism and Disposition.

[13]  Thomas Hartung,et al.  Chemical regulators have overreached , 2009, Nature.

[14]  Yves Grandvalet,et al.  Integrated Proteomic and Transcriptomic Investigation of the Acetaminophen Toxicity in Liver Microfluidic Biochip , 2011, PloS one.

[15]  Laurent Griscom,et al.  Behavior of HepG2/C3A cell cultures in a microfluidic bioreactor , 2011 .

[16]  Robert Combes,et al.  Toxicity testing: creating a revolution based on new technologies. , 2005, Trends in biotechnology.

[17]  M. Toner,et al.  Microfabricated grooved substrates as platforms for bioartificial liver reactors. , 2005, Biotechnology and bioengineering.

[18]  L. Samson,et al.  A microscale in vitro physiological model of the liver: predictive screens for drug metabolism and enzyme induction. , 2005, Current drug metabolism.

[19]  F. Bois,et al.  Zonation related function and ubiquitination regulation in human hepatocellular carcinoma cells in dynamic vs. static culture conditions , 2012, BMC Genomics.

[20]  Cécile Legallais,et al.  Metabolomics-on-a-chip and predictive systems toxicology in microfluidic bioartificial organs. , 2012, Analytical chemistry.

[21]  Yau Yi Lau,et al.  Development of a novel in vitro model to predict hepatic clearance using fresh, cryopreserved, and sandwich-cultured hepatocytes. , 2002, Drug metabolism and disposition: the biological fate of chemicals.

[22]  L. Goodman,et al.  The Pharmacological Basis of Therapeutics , 1941 .

[23]  D. Meijer,et al.  Drug traffic in the hepatobiliary system. , 1996, Journal of hepatology.

[24]  Paavo Honkakoski,et al.  Inhibition and induction of human cytochrome P450 enzymes: current status , 2008, Archives of Toxicology.

[25]  Don R. Maszle,et al.  MCSim: A Monte Carlo Simulation Program , 1997 .

[26]  M. Rissel,et al.  Use of hepatocyte cultures for the study of hepatotoxic compounds. , 1997, Journal of hepatology.

[27]  J Brian Houston,et al.  Prediction of in vitro intrinsic clearance from hepatocytes: comparison of suspensions and monolayer cultures. , 2005, Drug metabolism and disposition: the biological fate of chemicals.

[28]  E. Leclerc,et al.  Metabolomics-on-a-chip and metabolic flux analysis for label-free modeling of the internal metabolism of HepG2/C3A cells. , 2012, Molecular bioSystems.

[29]  Helmut Greim,et al.  Toxicological comments to the discussion about REACH , 2006, Archives of Toxicology.

[30]  W. Stigelman,et al.  Goodman and Gilman's the Pharmacological Basis of Therapeutics , 1986 .

[31]  J. Houston,et al.  PREDICTION OF METABOLIC CLEARANCE USING CRYOPRESERVED HUMAN HEPATOCYTES: KINETIC CHARACTERISTICS FOR FIVE BENZODIAZEPINES , 2005, Drug Metabolism and Disposition.

[32]  J Brian Houston,et al.  Evaluation of Cryopreserved Human Hepatocytes as an Alternative in Vitro System to Microsomes for the Prediction of Metabolic Clearance , 2007, Drug Metabolism and Disposition.

[33]  L. Griffith,et al.  Functional behavior of primary rat liver cells in a three-dimensional perfused microarray bioreactor. , 2002, Tissue engineering.

[34]  P. Beaune,et al.  Biochemical and analytical development of the CIME cocktail for drug fate assessment in humans. , 2010, Rapid communications in mass spectrometry : RCM.

[35]  Robert J Riley,et al.  EVALUATION OF FRESH AND CRYOPRESERVED HEPATOCYTES AS IN VITRO DRUG METABOLISM TOOLS FOR THE PREDICTION OF METABOLIC CLEARANCE , 2004, Drug Metabolism and Disposition.

[36]  Melvin E. Andersen,et al.  The need for a new toxicity testing and risk analysis paradigm to implement REACH or any other large scale testing initiative , 2006, Archives of Toxicology.

[37]  L. Griffith,et al.  Tissue Engineering--Current Challenges and Expanding Opportunities , 2002, Science.

[38]  N. Hewitt,et al.  Studies comparing in vivo:in vitro metabolism of three pharmaceutical compounds in rat, dog, monkey, and human using cryopreserved hepatocytes, microsomes, and collagen gel immobilized hepatocyte cultures. , 2001, Drug metabolism and disposition: the biological fate of chemicals.

[39]  Leslie M. Tompkins,et al.  Mechanisms of cytochrome P450 induction , 2007, Journal of biochemical and molecular toxicology.