In Vitro PK Studies in a Long-Term Liver Model
Tuesday, December 5, 2017 - 7:00AM PST, 10:00AM EST, 4:00PM CEST
Presenter: Dr. Nicole Kratochwil - Roche Innovation Center, Basel, Switzerland
Abstract: Long-term in vitro liver models are now widely explored for human hepatic metabolic clearance prediction, enzyme phenotyping, cross-species metabolism comparison of low clearance drugs as well as induction studies. Here, we present studies comparing different in vitro liver models (HepG2, iPSC-derived hepatocytes, HepaRG™ and HepatoPac®) with respect to their key metabolic activities. Similar metabolic activities were found for the long-term models, HepaRG™ and HepatoPac®, and the short-term suspension cultures when averaged across all 11 enzyme markers, although differences were seen in the activities of CYP2D6 and non-CYP enzymes. HepatoPac® was further evaluated with respect to clearance prediction. To assess the in vitro parameters, pharmacokinetic modeling was applied. The determination of intrinsic clearance by nonlinear mixed-effects modeling in a long-term model significantly increased the confidence in the parameter estimation and extended the sensitive range towards 3% of liver blood flow, i.e., >10-fold lower as compared to suspension cultures. The micropatterned model gave rise to clearance prediction in man within a two fold error for the majority of low-clearance compounds. In addition to clearance prediction, we present how metabolism, active transport, drug-drug interactions and induction may be assessed simultaneously as multiple endpoints in a single in vitro system. Thus, long-term liver models have great potential as translational research tools exploring pharmacokinetics of novel drugs in vitro.
Application of a micropatterned co-cultured (MPCC) hepatocyte system to predict preclinical and human specific drug metabolism
Wednesday, September 7, 2016 - 2:00 pm ET
Presenter: Dr. T. Eric Ballard - Pfizer
Summary: Laboratory animal models are the industry standard for use in preclinical risk assessments for drug candidates. Thus, it is important that these species possess profiles of drug metabolites that are similar to that anticipated in human, since metabolites could also be responsible for biological activities or unanticipated toxicity. Under most circumstances, preclinical species reflect human in vivo metabolites well; however, there have been several notable exceptions, and understanding and predicting these exceptions with an in vitro system would be very useful. Human micropatterned co-cultured (MPCC) hepatocytes have been shown to recapitulate human in vivo qualitative metabolic profiles but this had not been performed yet for laboratory animal species. In this study, we investigated several compounds that are known to produce human unique metabolites through CYP2C9, UGT1A4, aldehyde oxidase (AO) or N-acetyltransferase (NAT) that were poorly covered or not detected at all in the selected preclinical species. To perform our investigation we used 24-well MPCC hepatocyte plates having three individual human donors, a monkey, a dog and a rat donor to study drug metabolism at four time points per species. Through the use of the multi-species MPCC hepatocyte system, the metabolite profiles of the selected compounds with human donors effectively captured the qualitative in vivo metabolite profile with respect to the human metabolite of interest. Human unique metabolites that were not detected in vivo in certain preclinical species (normally dog and rat) were also not generated in the corresponding species in vitro confirming that the MPCC hepatocytes can provide an assessment of preclinical species metabolism. From these results, we conclude that multi-species MPCC hepatocyte plates could be used as an effective in vitro tool for understanding preclinical species metabolism relative to humans and aid in choosing the appropriate preclinical models.
SOLVO Webinar: Using micropatterned co-cultures to model human-specific drug metabolism, disposition and drug-drug interactions.
Thursday, March 10, 2016 - 10:00 am ET
Presenter: Dr. Salman Khetani - University of Illinois at Chicago
Summary: Since the liver metabolizes ~70% of marketed drugs, accurate prediction of liver’s role in the metabolism and disposition of drugs is a critical exercise in the drug development pipeline in order to flag compounds with poor pharmacokinetic characteristics and/or properly investigate liver-drug interactions before clinical trials. Animal models are not sufficient for such purposes given the differences between animals and humans in drug metabolism pathways. Thus, human-relevant assays are needed to supplement animal testing. Isolated primary human hepatocytes are widely considered to be the “gold standard” for creating human liver models; however, in culture formats that rely exclusively on extracellular matrix, hepatocyte functions (i.e. cytochrome P450s) display a rapid decline in key functions (i.e. drug metabolism enzymes, transporters), which prevents long-term drug dosing. In our view and others, an ideal hepatocyte culture platform should maintain high levels of drug metabolism enzymes with proper hepatocyte polarity for prolonged times in order to allow incubations with drugs that interact with multiple pathways, including transporters. Furthermore, such a culture platform should be compatible with multiple batches of cryopreserved hepatocytes for on-demand and reproducible screening, thereby avoiding inter-donor variability when necessary. Since primary human hepatocytes are a limited resource, a culture platform should also use as few hepatocytes as possible to allow screening over many more experiments than possible with fully confluent monolayers. The ability to evaluate drug disposition in MPCCs created using hepatocytes from different species (i.e. mouse, rat, dog, monkey, human) allows for selection of the most appropriate animal species for downstream in vivo investigations. Case studies from multiple pharmaceutical companies will be presented to demonstrate the “real-world” utility of the MPCC platform. More recently, we are creating versions of MPCCs that display specific pathologies (i.e. hepatitis, diabetes) towards ultimately investigating how drug disposition is affected with specific disease backgrounds. In the future, MPCCs can be used to reduce drug attrition and prevent harm to patients in the clinic.
HepatoPac®: Predicting clinically-relevant drug disposition and drug-drug interactions using micropatterned co-cultures.
Presented: November 2015
Presenter: Dr. Salman Khetani - University of Illinois at Chicago
Abstract: Metabolism by the liver accounts for the overall clearance of ~70% of marketed drugs. Thus, accurate prediction of in vivo human drug clearance using in vitro hepatic clearance data can help identify compounds with poor pharmacokinetic characteristics. An ideal culture platform for such purposes uses as few limited primary hepatocytes as possible in a reproducible/miniaturized format; maintains high levels of drug metabolism enzymes with proper hepatocyte polarity to allow incubations with drugs that interact with multiple pathways, including transporters; is compatible with multiple cryopreserved hepatocyte donors for on-demand screening; and, can be used to predict clearance of compounds with a wide range of turnover rates, including slowly metabolized compounds (i.e. low turnover). Additionally, the ability to interrogate effects of drug incubations on hepatocyte enzyme levels and subsequently victim drug disposition is important for modeling clinical drug-drug interactions. In this presentation, I will describe how micropatterned co-cultures (MPCCs) containing cryopreserved hepatocytes in multiwell plates approximate the aforementioned features and thus have been shown to be useful for CYP phenotyping, drug clearance prediction, metabolite identification and to model clinically-relevant drug-drug interactions. The ability to evaluate drug disposition in MPCCs created using hepatocytes from different species (i.e. mouse, rat, dog, monkey, human) allows for selection of the most appropriate animal species for downstream in vivo investigations. We are now creating versions of MPCCs that display specific pathologies (i.e. hepatitis, diabetes) towards ultimately investigating how drug disposition is affected with specific disease backgrounds. In the future, MPCCs can be used to reduce drug attrition and prevent harm to patients in the clinic.
HepatoMune™: A Hepatocyte-Kupffer Cell Co-Culture Model for Assessment of Pro-inflammatory Cytokine Effects on Metabolizing Enzymes and Drug Transporters.
Presented: April, 2015
Presenter: Dr. Onyi Irrechukwu - Hepregen Corporation
Based on a paper published with Merck.
Abstract: Alterations in the physiological levels of cytokines may be caused by inflammation, infection or drug administration/therapy. Therapeutic proteins (TPs), which are usually cytokines or cytokine modulators are being used increasingly in the clinic, and often in combination with small molecule drugs. Thus, there is a growing need for reliable in vitro models that can be used to investigate the effects of TPs on drug transporters and metabolizing enzymes and as such, on the disposition of small molecule drugs. Conventional cultures that are being used to study these cytokine effects are inherently limited because of the rapid decline in hepatocyte function and the absence of cell types that mediate the cytokine interactions with hepatocytes.
We have established a functional, stable and long-living hepatocyte-Kupffer coculture model, HepatoMuneTM, which enables the assessment of long–term effects of cytokines on CYP450 enzymes and hepatic transporters. We demonstrate that the HepatoMune platform is functional for up to 14 days in culture by evaluating the gene expression of CD163 (a macrophage specific receptor). Here, we present and explicate the differential effects of cytokines – IL-2, IL-23, IL-1β and IL-6 – on CYP450 activity and mRNA expression in the cocultures. Furthermore, we show that the HepatoMune cocultures exhibit an inflammatory response when stimulated with LPS or IL-1β, resulting in an increased release and up-regulation of acute phase proteins and other pro-inflammatory cytokines, and in greater suppression of metabolic enzyme and hepatic transporter gene expression compared to hepatocyte-only cultures. CYP suppression was preventable by blocking cytokine-receptor interaction using a receptor antagonist or monoclonal antibody against cytokine.
Thus, the HepatoMune platform, with the inclusion of a component of the innate immune system, is more physiologically relevant than conventional cultures, mediating interactions from a broader spectrum of cytokines which lack receptors on hepatocytes, and it may serve as a better predictive tool for assessing TP-DDI risks.
Hormone and Drug-mediated Modulation of Glucose Metabolism in a Microscale Model of the Human Liver
Presented: February 2015
Presenter: Dr. Salman Khetani - Colorado State University
Abstract: Due to its central role in glucose homeostasis, the liver is an important target for drug development efforts for type 2 diabetes mellitus (T2DM). Significant differences across species in liver metabolism necessitate supplementation of animal data with assays designed to assess human-relevant responses. However, isolated primary human hepatocytes (PHHs) display a rapid decline in phenotypic functions in conventional monolayer formats. Co-cultivation of PHHs with specific stromal cells, especially in micropatterned configurations, can stabilize some liver functions for ~4 weeks in vitro. However, it remains unclear whether co-culture approaches can stabilize glucose metabolism that can be modulated with hormones in PHHs. Thus, here we compared commonly employed conventional culture formats and previously developed micropatterned co-cultures (MPCCs) of cryopreserved PHHs and stromal fibroblasts for mRNA expression of key glucose metabolism genes (i.e. PCK1), and sensitivity of gluconeogenesis to prototypical hormones, insulin and glucagon. We found that only MPCCs displayed high expression of all transcripts tested for at least 2 weeks, and robust gluconeogenesis with responsiveness to hormones for at least 3 weeks in vitro. Furthermore, MPCCs displayed glycogen storage and lysis, which could be modulated with hormones under the appropriate feeding and fasting states, respectively. Finally, we utilized MPCCs in proof-of-concept experiments where we tested gluconeogenesis inhibitors and evaluated the effects of stimulation with high levels of glucose as in T2DM.
Phenotypic Profiling Strategies using High-throughput High Content Imaging Approaches to Examine the Outcome of Compound Challenge in Primary Human Hepatocytes
Presented: January 2015
Presenter: Joe Trask - Hamner Institutes for Health Sciences
Abstract: The promise of newer technologies like High Content Screening have provided researchers insights to de-convolute and better understand complex biological pathways and processes. Researchers may now utilize not only in vitro cell models, but also ex vivo and in vivo models to study the outcome following exposure to drugs and chemicals. In this presentation I will provide the audience with a brief background of high content imaging technologies strategies for developing robust assays for compound discovery and profiling, including key references, and examples of how we have adopted this technology and are using it to investigate the effects of compound challenge in an in vitro cell model.
Microscale Engineering Strategies to Functionally Mature iPSC-derived Human Hepatocytes.
Presented: September, 2014
Presenters: Dr. Salman Khetani - Colorado State University, David Mann - Cellular Dynamics
Abstract: Induced pluripotent stem cells (iPSCs) have near infinite replication potential and can be differentiated into cell types from all three germ layers of the body in vitro. In the case of the liver, induced pluripotent stem cell-derived human hepatocytes (iHeps), commercially available from Cellular Dynamics as iCell® Hepatocytes, could provide a complementary tool to primary cells for studying the mechanisms underlying human liver development and disease, testing the efficacy and safety of pharmaceuticals across different patients (i.e. personalized medicine), and enabling cell-based therapies in the clinic. However, current in vitro protocols that utilize growth factors and extracellular matrices (ECM) alone yield iHeps with fetal levels of liver functions relative to adult primary human hepatocytes (PHHs). Furthermore, these low hepatic functions in iHeps are difficult to maintain for prolonged times in culture. We have engineered a micropatterned co-culture (iMPCC) platform in a multi-well format (24- and 96-well plates) that significantly enhanced the functional maturation and longevity of fresh and cryopreserved iHeps in culture for at least 4 weeks in vitro when compared to standard confluent cultures. In particular, iHeps were micropatterned onto ECM-coated domains of empirically optimized dimensions using soft lithographic techniques and subsequently surrounded by supportive 3T3-J2 murine embryonic fibroblasts. Overlaying the iMPCCs with an ECM gel further improved iHep functions. We assessed iHep maturity via liver gene expression (i.e. HNF4a), secretion of albumin and urea, basal CYP450 activities, phase II conjugation, drug-mediated CYP450 induction, and hepatocyte polarity (i.e. canalicular transport). Moreover, we showed for the first time that the predictive power for classifying drugs as liver-toxic or non-toxic in iMPCCs was remarkably similar to stable cultures of PHHs, thereby demonstrating iHep utility in early stages of drug development where a paucity of healthy human liver tissues often limits PHH use for testing drug-induced liver toxicity. In the future, iMPCCs could provide a more mature and long-term culture platform for studying molecular mechanisms underlying iHep differentiation, modeling liver diseases, and integration into organs-on-a-chip systems being designed to assess multi-tissue responses to compounds and other perturbations.
Modeling Hepatotropic Infections in Human Micropatterned Co-Cultures
Presented July 2014
Presenter: Dr. Sangeeta Bhatia - MIT
Abstract: Human pathogens are responsible for over half of human deaths each year. The evolution of disease pathogenesis is dependent on complex host-pathogen interactions, however, model systems that replicate the pathogen life-cycle and capture these interactions are often lacking. Several important pathogens are restricted to infect the liver and are further species-restricted to human hepatocytes including: hepatitis A,B,C and D viruses as well as the human malarias. We have developed a microscale liver platform consisting of primary human hepatocytes, micropatterned and surrounded by stromal cells (MPCC) that maintains a differentiated hepatocyte phenotype for weeks and supports the complete viral life cycle of hepatitis C virus, enabled the development and study of the liver stages of human malaria (Plasmodium falicparum and vivax), and demonstrated that hepatocytes are not mere bystanders but robustly respond to hepatotropic infection. Thus, MPCC offer new opportunities for the study of human liver disease and host-pathogen interactions that may inform the development of therapeutic interventions.
HepatoMune™: An in vitro model for studying inflammation-drug interactions
Presented June 2014
Presenter: Dr. Onyi Irrechukwu - Hepregen
Abstract: The co-occurrence of inflammation and drug therapy or drug exposure has been shown to reduce the threshold for drug toxicities, leading to drug-induced hepatotoxicity. Inflammation has also been shown to alter the activities of enzymes and transporters involved in drug metabolism. Physiologically, inflammatory episodes can be caused by several factors including xenobiotics/drugs, infection, and disease. Therefore, co-administration of drugs demands special considerations due to potential pharmacological and toxicological consequences. An in vitro model that mimics liver inflammation may provide better predictive data in preclinical testing of xenobiotics. In this webinar, I will discuss the development of the HepatoMune tri-culture platform by supplementing Hepregen's long-living HepatoPac platform with primary Kupffer macrophages. I will also discuss the criteria that were evaluated to ensure we had an optimally functioning platform. Finally, I will present data showing the use of the HepatoMune platform to detect toxicities exacerbated and/or precipitated by inflammation-drug interactions. At the end of this webinar, the audience will gain insight into the utility of the HepatoMune platform for predicting drug induced liver injury mediated by inflammatory stress.
The Micropatterned Co-culture Platform: Past, Present and the Future.
Presented May 2014
Presenter: Dr. Salman Khetani - Colorado State University
Abstract: Micropatterned co-cultures (MPCC) were first developed more than 20 years ago to test hypotheses around how controlled homotypic and heterotypic cell-cell interactions affect rat hepatocyte functions in vitro. Since the first proof-of-concept, the MPCC platform has evolved into one of the most functionally stable and long-term models of the liver, containing primary hepatocytes from either animal (rat, mouse, dog, monkey) or human species arranged in empirically optimized clusters and surrounded by stromal fibroblasts. In this presentation, I will first discuss the design principles behind the MPCC platform including the importance of architecture in engineering functionally optimal co-cultures. Then, I will present both published and unpublished data sets on key applications relevant to the pharmaceutical industry such as drug metabolism, toxicology and disease modeling where MPCCs have shown clear advantages over standard hepatocyte culturing techniques. Finally, applications of the MPCC model to liver diseases (such as hepatitis C virus, malaria and type 2 diabetes), as well as use of iPSC-derived human hepatocytes in MPCCs will be discussed as next generation and forward looking approaches. At the end of this presentation, the audience will gain insights into the design decisions underlying the MPCC platform, its main advantages for drug screening and how it can add predictive value to applications relevant to the pharmaceutical industry.
Application of HepatoPac® Toward Developing an Integrated Understanding of the Disposition and Metabolism of Faldaprevir.
Presented April 2014
Presenter: Diane Ramsden - Boehringer Ingelheim
Abstract: Surprises happen all of the time in Drug Development. For example, there are numerous cases of 'novel' metabolites being discovered in human 14[C] ADME studies, either due to the use of radiolabel or metabolites found in excreta that do not circulate to any great extent.
With faldaprevir, the human 14[C] ADME data indicated that there were two major hydroxylated metabolites found in excreta accounting for almost half of the dose of parent drug, the other major component being unchanged parent. All of the pre-existing data (in vitro and in vivo), including clinical studies, indicated that faldaprevir was a low clearance compound with metabolism being a minor contributor of overall clearance. Suddenly a number of questions arose.
Where are the metabolites formed? Why aren't they observed in circulation? Why weren't they observed to an appreciable level in in vitro test systems or in pre-clinical models? Is there coverage in the pre-clinical species? Do they contribute to efficacy? Can the formation and elimination pathways be identified? How does unchanged parent get into excreta? Is it unabsorbed or does it undergo biliary excretion? Is there an acyl-glucuronide metabolite formed and cleaved and does this become a major elimination pathway that should be further explored?
This webinar will focus on how these questions were answered by applying a novel, long-term, co-culture hepatocyte model, HepatoPac. Topics covered will include, (a) validation of the human HepatoPac model using rat HepatoPac to provide an IVIVC, (b) application of human HepatoPac to delineate the importance of transporter and enzyme interplay, and (c) insights into the formation, disposition and elimination of faldaprevir to provide an integrated model of faldaprevir clearance.
Utility of Micropatterned Co-Cultures for Drug Toxicity Studies.
Presented November 2013
Presenter: Dr. Salman Khetani - Colorado State University
Abstract: Micropatterned co-cultures (MPCC) were developed as a more stable and long-term model of the liver, containing primary hepatocytes from animal (rat, mouse, dog, monkey) and human species arranged in empirically optimized clusters and surrounded by 3T3-J2 murine embryonic fibroblasts. The utility of this model system has been demonstrated in several applications relevant for drug development, including, but not limited to, drug clearance prediction, drug metabolite identification, hepatitis C infection, malaria, enzyme induction and more recently, type 2 diabetes. In this presentation, Dr. Khetani will discuss the application of MPCCs for drug toxicity prediction, both in a screening/selection mode as well as to understand mechanism of action underlying the toxicity. In particular, data from a 64 compound screening set will be presented with respect to the sensitivity and specificity of MPCCs (rat and human) as compared to clinical outcomes and conventional sandwich cultures. Additionally, a case study of acetaminophen bio-activation will be presented to demonstrate how MPCCs can be used to understand specific mechanisms by which toxins cause hepatic injury. Finally, applications of the MPCC model to liver diseases such as hepatitis C and type 2 diabetes, as well as use of iPSC-derived human hepatocytes in MPCCs will be discussed as next generation and forward looking approaches.
The Development and Validation of HepatoPac®, a Next Generation In Vitro Microliver Model.
Presented July 2013
Presenter: Dr. Sangeeta Bhatia - MIT
Abstract: Primary hepatocytes display a precipitous decline in phenotypic functions using conventional models such as sandwich cultures with extracellular matrix proteins (i.e. collagen, Matrigel). This severely limits the ability of these model systems to accurately predict clinical outcomes. HepatoPac® technology extends culture stability and sensitivity by organizing hepatocytes into colonies surrounded by supportive, non-parenchymal cells. HepatoPac® co-cultures result in a high-fidelity, in vitro tissue model that is proven to provide a range of liver-specific functions for greater than 4 weeks.
PBS, Nova Channel.
Hear from Sangeeta Bhatia, an MIT biomedical engineer and inventor of HepatoPac® technology.