Chan T.S.,Boehringer Ingelheim Pharmaceuticals |
Yu H.,Boehringer Ingelheim Pharmaceuticals |
Moore A.,Hepregen, Inc. |
Khetani S.R.,Colorado State University |
Tweedie D.,Boehringer Ingelheim Pharmaceuticals
Drug Metabolism and Disposition | Year: 2013
Generating accurate in vitro intrinsic clearance data is an important aspect of predicting in vivo human clearance. Primary hepatocytes in suspension are routinely used to predict in vivo clearance; however, incubation times have typically been limited to 4-6 hours, which is not long enough to accurately evaluate the metabolic stability of slowly metabolized compounds. HepatoPac is a micropatterened hepatocyte-fibroblast coculture system that can be used for continuous incubations of up to 7 days. This study evaluated the ability of human HepatoPac to predict the in vivo clearance (CL) of 17 commercially available compounds with low to intermediate clearance (<12 ml/min per kg). In vitro half-life for disappearance of each compound was converted to hepatic clearance using the well stirred model, with and without correction for plasma protein binding. Hepatic CL, using three individual donors, was accurately predicted for 10 of 17 compounds (59%; predicted clearance within 2-fold of observed human in vivo clearance values). The accuracy of prediction increased to 76% (13 of 17 compounds) with an acceptance criterion defined as within 3-fold. When considering only low clearance compounds (<5 ml/min per kg), which represented 10 of the 17 compounds, the accuracy of prediction was 60% within 2-fold and 90% within 3-fold. In addition, the turnover of three slowly metabolized compounds (alprazolam, meloxicam, and tolbutamide) in HepatoPac was directly compared with turnover in suspended hepatocytes. The turnover of alprazolam and tolbutamide was approximately 2-fold greater using HepatoPac compared with suspended hepatocytes, which was roughly in line with the extrapolated values (correcting for the longer incubation time and lower cell number with HepatoPac). HepatoPac, but not suspended hepatocytes, demonstrated significant turnover of meloxicam. These results demonstrate the utility of HepatoPac for prediction of in vivo hepatic clearance, particularly with low clearance compounds. Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics.
Lin C.,Colorado State University |
Lin C.,University of Illinois at Chicago |
Shi J.,Hepregen, Inc. |
Moore A.,Hepregen, Inc. |
And 2 more authors.
Drug Metabolism and Disposition | Year: 2016
Accurate prediction of in vivo hepatic drug clearance using in vitro assays is important to properly estimate clinical dosing regimens. Clearance of low-turnover compounds is especially difficult to predict using short-lived suspensions of unpooled primary human hepatocytes (PHHs) and functionally declining PHH monolayers. Micropatterned cocultures (MPCCs) of PHHs and 3T3-J2 fibroblasts have been shown previously to display major liver functions for several weeks in vitro. In this study, we first characterized long-term activities of major cytochrome P450 enzymes in MPCCs created from unpooled cryopreserved PHH donors. MPCCs were then used to predict the clearance of 26 drugs that exhibit a wide range of turnover rates in vivo (0.05-19.5 ml/min per kilogram). MPCCs predicted 73, 92, and 96% of drug clearance values for all tested drugs within 2-fold, 3-fold, and 4-fold of in vivo values, respectively. There was good correlation (R2 = 0.94, slope = 1.05) of predictions between the two PHH donors. On the other hand, suspension hepatocytes and conventional monolayers created from the same donor had significantly reduced predictive capacity (i.e., 30-50% clearance values within 4-fold of in vivo), and were not able to metabolize several drugs. Finally, we modulated drug clearance in MPCCs by inducing or inhibiting P450s. Rifampin-mediated CYP3A4 induction increased midazolam clearance by 73%, while CYP3A4 inhibition with ritonavir decreased midazolam clearance by 79%. Similarly, quinidine-mediated CYP2D6 inhibition reduced clearance of dextromethorphan and desipramine by 71 and 22%, respectively. In conclusion, MPCCs created using cryopreserved unpooled PHHs can be used for drug clearance predictions and to model drug-drug interactions. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.
Trask O.J.,Hamner Institutes for Health Sciences |
Moore A.,Hepregen, Inc. |
Lecluyse E.L.,Hamner Institutes for Health Sciences
Assay and Drug Development Technologies | Year: 2014
The current landscape of in vitro models used to identify drug-or chemical-induced hepatotoxicity relies heavily on cell culture models consisting of HepG2, induced pluripotent stem cell-derived, or primary hepatocytes. While these in vitro models offer powerful approaches for predicting toxicity, each system has challenges, including variable metabolic capacity, brief ex vivo life span in culture, and adoption with standard automated microscopy high-content screening (HCS) systems to measure reproducibility data at the single-cell level. In this report we introduce a novel primary hepatocyte coculture model, HepatoPac™, as an alternative to current model systems for evaluation of in vitro hepatotoxicity in 96-well microtiter plate format examined by HCS. The coculture model consists of primary hepatocytes that are micropatterned to form a discrete microarchitecture or "hepatocyte islands" that are surrounded by supporting fibroblasts resulting in long-term viability and metabolic function of primary hepatocytes. Using multiple HCS image capture and image analysis strategies, we established methods to interrogate various morphometric parameters, such as size, shape, and intensity, at the island or single-cell level. We applied these approaches to identify subpopulations of both fibroblasts and hepatocytes that exhibited alterations in nuclear parameters, cell permeability, mitochondria function, and apoptosis using known reference control compounds and an eight-point dose curve. Subpopulation analysis with additional bioprobe sets can provide a powerful means of addressing differential cell and tissue susceptibilities during compound profiling. Our data show that the HepatoPac is amendable for HCS imaging applications and provides a unique approach for studying hepatotoxicity over prolonged periods of time. © 2014, Mary Ann Liebert, Inc.
Hepregen, Inc. | Date: 2013-03-12
The present disclosure provides compositions, systems, and tools for modeling liver inflammation and methods of using the same. The disclosure provides micropatterned hepatocyte co-cultures where individual cell populations remain functionally stable during long-term culture. The in vitro liver inflammation models of the present disclosure may be useful for evaluating inflammation-mediated toxicities of compounds in a pre-clinical setting.
Hepregen, Inc. | Date: 2016-01-08
The present disclosure provides compositions, systems, and tools for modeling liver inflammation and methods of using the same. The disclosure provides micropatterned hepatocyte-Kupffer cell co-cultures or Kupffer cell-treated hepatocyte co-cultures where hepatocytes maintain Kupffer cell-initiated activities during long-term culture. The in vitro liver inflammation models of the present disclosure may be useful for evaluating both acute as well as chronic inflammation-mediated toxicities of compounds in a pre-clinical setting.
Agency: National Science Foundation | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 500.00K | Year: 2010
This Small Business Innovation Research (SBIR) Phase II project is aimed towards development of a human micro-liver platform and assay technologies for cost-effective, high-throughput, and quantitative screening of drug-induced liver injury (DILI) and drug-drug interactions (DDI) following chronic exposure to pharmaceuticals. While primary human hepatocytes isolated from the liver are widely utilized in the pharmaceutical industry for drug screening, these cells rapidly (hours) lose phenotypic functions under conventional culture conditions. Recently, a human liver tissue model with defined microscale architecture has been developed that maintains phenotypic functions of primary hepatocytes for several weeks in vitro (micro-livers). This project proposes to develop assay technologies (gene expression, reporter-based, and high content imaging) with micro-livers in a high-throughput multi-well format for DILI and DDI screening in early drug discovery. The broader impacts of this research are novel approaches for the development of high-throughput, physiologically-relevant platforms for assessing the potential of compounds to cause adverse effects on organs. The liver platforms developed here may enable the elimination of drugs with problematic toxicity profiles much earlier in the drug development pipeline towards substantially reducing the cost to develop a successful drug ($1 billon per drug), increasing the likelihood of clinical success, and limiting human exposure to unsafe drugs. In the future, these platforms may be useful for evaluating the injury potential of environmental toxicants, in fundamental investigations of liver physiology and disease, and for personalized medicine.
Hepregen, Inc. | Date: 2011-01-21
Systems and methods for using stamps to print or mask materials on a substrate service. In one particular embodiment, the systems and methods include a microcontacting stamp that has a plurality of rigid posts each having a resilient pad at its distal end. Each post is fitted within an aperture located in a guide plate such that the post may move longitudinally within the guide plate. The guide plate includes a variety of apertures that typically are aligned with the wells of a microtiter plate. The apertures extend typically through the entire thickness of the guide plate. On one side of the guide plate is a resilient member that extends over one or more of the apertures thereby holding the post in place.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 113.04K | Year: 2010
DESCRIPTION (provided by applicant): Liver toxicity is the leading cause of pre-launch and post-market attrition of pharmaceutical compounds (i.e. Rezulin, Prexige). Significant species-specific differences in organ functions now necessitate supplementation of animal data with assays designed to assess human responses to drugs. Adherent cultures of primary human hepatocytes are considered to be the gold standard for evaluating preclinical drug metabolism, enzyme induction, and liver-specific toxicity. However, hepatocytes display a rapid (hours) decline in liver-specific functions under conventional culture conditions utilized routinely for drug development. Recently, a robust model of human liver tissue has been developed with optimized microscale architecture in an industry-standard multiwell format that retains liver-specific functions for 4-6 weeks in vitro. Hepatocytes in this microscale platform secrete liver-specific products, display functional CYP450 and conjugation enzymes, secrete molecules into the bile canaliculi, and maintain high levels of expression of liver-specific genes relevant for evaluating drug disposition. The primary objective of this Small Business Innovation Research (SBIR) Phase II project is to further develop and optimize these microscale human liver cultures and couple them with miniaturization strategies and assay technologies for cost-effective high-throughput in vitro screening. Since drug-induced liver injury (DILI) is a leading cause of acute liver failures and the high attrition rate of pharmaceuticals, we will optimize our miniaturized human livers specifically for the in vitro screening of genotype-specific and clinically-relevant drug disposition and coupled DILI. The technologies we develop here may find broad utility in the development of several classes of therapeutic compounds (drugs, biologics), in evaluating the disposition and injury potential of environmental toxicants, in fundamental investigations of liver physiology and disease, in the identification of new biomarkers, and in personalized medicine for liver disease. In the future, continued combination of microtechnology with tissue engineering may spur the development of other tissue models and their integration into the so-called 'human-on-a-chip'. PUBLIC HEALTH RELEVANCE: The studies proposed in this project are aimed towards developing a miniaturized human liver microarray for high-throughput screening (HTS) applications, specifically for evaluating drug disposition and drug-induced liver injury, a serious challenge for patients, regulatory agencies and the pharmaceutical/biotech industry. In the future, our miniaturized micro-liver HTS system may eliminate problematic compounds much earlier in the drug development pipeline towards reducing patient exposure to unsafe drugs. The technologies we develop here may also find utility in assessing the injury potential of environmental toxicants, in basic research, and in personalized medicine for patients with liver disease.
Hepregen, Inc. | Date: 2016-01-26
The present disclosure provides compositions, systems, and tools for modeling the canine liver and methods of using the same. The disclosure provides micropatterned hepatocyte co-cultures where individual cell populations remain functionally stable during long-term culture.