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COLUMBIA, MD, United States

Human hepatocytes, with complete hepatic metabolizing enzymes, transporters and cofactors, represent the gold standard for in vitro evaluation of drug metabolism, drug-drug interactions, and hepatotoxicity. Successful cryopreservation of human hepatocytes enables this experimental system to be used routinely. The use of human hepatocytes to evaluate two major adverse drug properties: drug-drug interactions and hepatotoxicity, are summarized in this review. The application of human hepatocytes in metabolism-based drug-drug interaction includes metabolite profiling, pathway identification, P450 inhibition, P450 induction, and uptake and efflux transporter inhibition. The application of human hepatocytes in toxicity evaluation includes in vitro hepatotoxicity and metabolism-based drug toxicity determination. A novel system, the Integrated Discrete Multiple Organ Co-culture (IdMOC) which allows the evaluation of nonhepatic toxicity in the presence of hepatic metabolism, is described. Source

Li A.P.,In Vitro Admet Laboratories, Llc
Biomarkers in Medicine | Year: 2014

The accuracy of preclinical safety evaluation to predict human toxicity is hindered by species difference in drug metabolism and toxic mechanism between human and nonhuman animals. In vitro human-based experimental systems allowing the assessment of human-specific drug properties represent a logical and practical approach to provide human-specific information. An advantage of in vitro approaches is that they require only limited amounts of time and resources, and, most importantly, do not invoke harm to human patients. Human hepatocytes, with complete hepatic metabolizing enzymes, transporters and cofactors, represent a practical and useful experimental system to assess drug metabolism. The use of human hepatocytes to evaluate two major adverse drug properties, drug-drug interactions and hepatotoxicity, are reviewed. The application of human hepatocytes in metabolism-based drug-drug interactions includes metabolite profiling, pathway identification, CYP450 inhibition, CYP450 induction, and uptake and efflux transporter inhibition. The application of human hepatocytes in toxicity evaluation includes in vitro hepatotoxicity and metabolism-based drug toxicity determination. Correlation of drug toxicity with proteomics and genomics data may allow the discovery of clinical biomarkers for early detection of liver toxicity. © 2014 Future Medicine Ltd. Source

Li A.P.,In Vitro Admet Laboratories, Llc
Current Topics in Pharmacology | Year: 2013

Successful cryopreservation of hepatocytes, especially human hepatocytes, is one of the major reasons for the recent routine application of this experimental system in drug development. Cryopreserved human hepatocytes retain viability and metabolic capacity and are used extensively as suspension cultures to evaluate the metabolic fate (metabolic stability and metabolite profiling) of new chemical entities (NCE) during drug development. Pooled cryopreserved human hepatocytes, i.e., hepatocytes cryopreserved from several individual donors that have been thawed, pooled, and re-cryopreserved, represent the most commonly used system for routine metabolism studies. One major issue with cryopreservation, namely, the loss of the ability of the cells to be cultured, has been overcome. Now hepatocytes from both animals and humans can be cryo- preserved to retain their ability to form monolayer cultures (known as " plateable " cryopreserved hepatocytes). The use of " plateable " cryopreserved hepatocytes enhances experimental efficiency by providing an immediate supply of easily stored cells and eliminating the centrifugation steps to remove test articles after treatment (e.g. uptake and time-dependent inhibition studies), which is required for suspension cultures. Plating extends their use in applications that involve culturing for a prolonged period (multiple days), such as evaluating metabolic stability of slowly-metabolized compounds, P450 induction, efflux transport, and hepatotoxicity. A significant advancement in the application of plateable cryopreserved hepatocytes is the evaluation of the role of metabolism-based drug toxicity on extrahepatic organs in a single test system, especially the novel Integrated Discrete Multiple Organ Co-culture (IdMOC™) system. Source

Li A.P.,In Vitro Admet Laboratories, Llc | Schlicht K.E.,Agilent Technologies
Drug Metabolism Letters | Year: 2014

A higher throughput platform was developed for the determination of KM values for isoform-selective P450 substrates in human hepatocytes via incubation of the hepatocytes with substrates in 384-well plates and metabolite quantification by RapidFire™ mass spectrometry. Isoform-selective P450 substrates were incubated at 8 concentrations in triplicate with cryopreserved human hepatocytes from 16 donors. The metabolic pathways examined were the CYP1A2-catalyzed tacrine 1 -hydroxylation, CYP2B6-catalyzed bupropion hydroxylation, CYP2C8-catalyzed amodiaquine N-deethylation, CYP2C9-catalyzed diclofenac 4'-hydroxylation, CYP2D6-catalyzed dextromethorphan O-demethylation, and CYP3A4-catalyzed midazolam 1'-hydroxylation. Typical saturation enzyme kinetics was observed for all the pathways evaluated. Individual differences in the apparent Vmax and KM values were observed among the human hepatocytes from each of the 16 individual donors, with no statistically significant gender- or age-associated differences. A “composite” KMvalue was calculated for each of the pathways via normalizing the individual activities to their respective Vmax values to develop “relative activities” followed by Michaelis-Menten analysis of the mean relative activities of the 16 donors at each of the 8 substrate concentrations. The resulting “composite” KM values for the P450 substrates may be used to guide in vitro P450 inhibition and induction studies and kinetic modeling of in vivo drug-drug interaction. © 2014 Bentham Science Publishers. Source

Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 100.00K | Year: 2011

DESCRIPTION (provided by applicant): The objective of our grant application is to develop an in vitro model of the blood-brain barrier and assays to examine permeability by using isolated brain microvessels. Unlike capillaries in other organs, brain capillaries establish a barrier that is largely impermeable to polar and larger chemicals, which are due to tight junctions, the absence of both fenestrae and pinocytosis. Although, the permeability of most chemicals to cross the blood-brain barrier is directly associated to its' oil/water partition coefficient, many lipophillic chemicals fail to enter the brain because of the expression of the multidrug transporters. In contrast, polar nutrients that would be predicted to be less permeable display higher uptake because of the expression of nutrient transporters. Many chemicals that are potentially toxic to the brain fail to cross the blood-brain barrier. Additionally, many drug candidates for treating mental illnesses and neurological diseases fail because ofthe blood-brain barrier. Consequently, a model to study the blood-brain barrier is needed for the new paradigm in which testing chemicals for toxicity will be accomplished with in vitro and in silico methods. Additionally, a model is needed to aid chemists in their attempts to design neurotrophic drugs. Current cell culture models fail to display the tightness and the many transporters found in vivo. In contrast, isolated brain microvessels retain all of the transporters and display a tight barrier, and have been used in the past 40 years in research studies on blood-brain barrier permeability. The major obstacles in using isolated brain microvessels have been the laborious procedure for their preparation and their short life span. A commercial source of brain microvessels will overcome these obstacles. The overall objective of our study is to develop cryopreserved BM as a model of the blood-brain barrier and optimize reagents and assays to measure permeability and transport that will either be conducted byour company or sold to others. To accomplish the objective, in Specific Aim 1 conditions will be standardized for preparing, cryopreserving viable bovine brain microvessels and assure consistency in different batches of microvessels sold. The functionalityof the brain microvessels will be assessed by conducting assays to measure amino acid and sugar transport, multidrug transporters, and viability. Assays achieving Z-factors closest to 1.0 will indicate the best preparation conditions. In Specific Aim 2, assays will be developed to examine chemical toxicity of the blood-brain barrier by measuring non specific transport. Sensitivity and specificity will be assessed by computing receiver operating curves. In the phase 2 grant application, assays will be developed to measure multi drug efflux pumps and the luminal and abluminal transporters. By establishing a commercial source of reagents for assessing the blood-brain barrier, pharmaceutical companies and chemical testing laboratories will have a readily available model to screen test chemicals for permeability and toxicity. When considering the importance of the blood-brain barrier in neurological functions, these products will potentially have wide distribution and commercial success. PUBLIC HEALTH RELEVANCE: The lack of an effective model for measuring the effects of drugs and chemicals on the blood-brain barrier (BBB) has been an impediment to evaluating chemicals for toxicity to the brain as well as to the successful development of drugs to treatdiseases of the brain18,29. The proposed model of the blood-brain barrier, isolated brain microvessels (BM) from cows, and assay for blood-brain barrier permeability, has the potential for enormous impact in the field of neurotoxicity testing and drug development. The innovation here is in taking a successful research model, freshly isolated BM, and converting it into a cryopreserved, marketable product as well as translating its use into assays that address the previously unmet need for evaluating BBB permeability.

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