COLUMBIA, MD, United States

In Vitro Admet Laboratories, Llc
COLUMBIA, MD, United States
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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.

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.

Yang Q.,In Vitro Admet Laboratories, Llc | Doshi U.,In Vitro Admet Laboratories, Llc | Li N.,In Vitro Admet Laboratories, Llc | Li A.P.,In Vitro Admet Laboratories, Llc
Current Drug Metabolism | Year: 2012

We report here a comprehensive evaluation of the effects of culture duration on the gene expression of P450 isoforms, uptake transporters and efflux transporters in human hepatocyte cultured in the absence and presence of the prototypical proinflammatory cytokine, interleukin-6 (IL-6). Primary collagen-matrigel sandwich cultures of human hepatocytes were cultured in supplemented William's E medium containing 0, 0.1, 0.5 and 5 ng/mL of IL-6 for the time periods of 2, 6, 12, 24 and 48 hrs. Real-time PCR was performed to quantify gene expression of acute phase proteins (suppressor of cytokine signaling 3 (SOCS-3), c-reactive protein (CRP) and lipopolysaccharide (LPS)-binding proteins (LBP)); P450 isoforms (CYPs 1A2, 2B6, 2C8, 2C9, 2D6, 3A4, and 3A5), uptake transporters (SLC10A1, SLC22A1, SLC22A7, SLCO1B1, SLCO1B3, SLCO2B1) and efflux transporters (ABCB1, ABCB11, ABCC2, ABCC3, ABCC4, ABCG2). SOCS-3, CRP, and LBP were extensively induced by IL-6, with maximal induction observed at 2 (SOCS-3) and 12 hrs (CRP; LBP), demonstrating that the cultured human hepatocytes responded to IL-6 treatment. In the untreated group (control), gene expression of P450 isoforms and uptake transporters decreased while efflux transporters remained relatively stable or increased with cultured duration. IL-6 predominantly caused down regulations of the genes studied, with the most significant changes observed at different treatment durations, apparently related to the stability of the basal levels of gene expression. For instance, for genes with unstable expression, which would decrease rapidly in culture (e.g CYP3A4), the most definitive down regulatory effects were observed at a relatively early time point (e.g. 12 hrs). In contrast, a longer treatment duration (e.g. 48 hrs) was required for genes with relatively stable expression levels in culture (e.g. ABCB1). Based on our findings, evaluation of multiple treatment durations rather than single treatment duration is recommended for the evaluation of biotherapeutics in cultured human hepatocytes where down regulation is expected. © 2012 Bentham Science Publishers.

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.

Li A.P.,In Vitro Admet Laboratories, Llc | Doshi U.,In Vitro Admet Laboratories, Llc
Drug Metabolism Letters | Year: 2011

Time-dependent or mechanism-based CYP3A4 inhibition is an important adverse drug property that should be carefully managed during drug development. Evaluation of time-dependent inhibition is traditionally performed using liver microsomes or recombinant P450 isoforms. We report here higher throughput approaches to evaluate time-dependent CYP3A4 inhibition assay using cultured cryopreserved human hepatocytes. The assays were performed in human hepatocytes cultured in 96-well plates, with luciferin-IPA as the CYP3A4 specific substrate. The advantages of the approach are as follows: 1. The use of 96-well plates minimizes the quantity of human hepatocytes and test materials required for the assays. 2. The use of luciferin-IPA allows CYP3A4 activity to be quantified rapidly using a plate reader, thereby avoiding the need for LC/MS that is required for traditional substrates such as testosterone and midazolam. 3. The use of cultured (plated) hepatocytes allows effective removal of treatment medium and washing of the cells without the laborious centrifugation step that is required for hepatocytes in suspension. Two assays were developed: 1. IC 50 shift assay; and 2. enzyme kinetic assay. The IC 50 shift assay is intended for general screening purpose with which a time-dependent CYP3A4 inhibitor would be identified by an increase in inhibitory potency (quantified as a decrease in IC 50) upon a 30 min. pre-incubation of hepatocytes with the inhibitor at 37 deg. C. Results with model inhibitors showed that the IC 50 assay readily distinguished the time-dependent inhibitors (1-aminobenzotriazole, erythromycin) from the non-timedependent inhibitor (ketoconazole). The enzyme kinetic assay is used for the derivation of the kinetic parameters K I and k inact. With this assay, time and concentration dependent inhibition of CYP3A4 were observed for 1-aminobenzotriazole and erythromycin. With hepatocytes from 4 donors, K I and k inact values were calculated to be 22.0 to 70.7 uM, and 0.09 to 0.51 min -1, respectively, for 1-aminobenzotriazole; and 47.3 to 75.1 uM, and 0.26 to 1.48, respectively, for erythromycin. DMSO (tested up to 2% v/v) was found to significantly attenuate the time-dependent inhibitory effects of 1- aminobenzotriazole, and had no apparent effects on erythromycin. Acetonitrile and methanol at 1% v/v had significantly less effects. The higher throughput assays describe here can to be used routinely for the evaluation of time-dependent CYP3A4 inhibitory potential of drug candidates during early phases of drug development. © 2011 Bentham Science Publishers.

Utkarsh D.,In Vitro Admet Laboratories, Llc | Loretz C.,In Vitro Admet Laboratories, Llc | Li A.P.,In Vitro Admet Laboratories, Llc
Chemico-Biological Interactions | Year: 2015

A possible risk factor for drug-induced hepatotoxicity is drug metabolizing enzyme activity, which is known to vary among individuals due to genetic (genetic polymorphism) and environmental factors (environmental pollutants, foods, and medications that are inhibitors or inducers of drug metabolizing enzymes). We hypothesize that hepatic cytochrome P450-dependent monooxygenase (CYP) activity is one of the key risk factors for drug induced liver injuries (DILI) in the human population, especially for drugs that are metabolically activated to cytotoxic/reactive metabolites. Human hepatocytes from 19 donors were evaluated for the activities of 8 major P450 isoforms: CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. Extensive individual variations were observed, consistent with what is known to be in the human population. As CYP3A4 is known to be one of the most important P450 isoforms for drug metabolism, studies were performed to evaluate the relationship between the in vitro cytotoxicity of hepatotoxic drugs and CYP3A4 activity. In a proof of concept study, hepatocytes from six donors (lots) representing the observed range of CYP3A4 activities were chosen for the evaluation of in vitro hepatotoxicity of four drugs known to be associated with acute liver failure: acetaminophen, cyclophosphamide, ketoconazole, and tamoxifen. The hepatocytes were cultured in collagen-coated plates and treated with the hepatotoxicants for approximately 24 h, followed by viability determination based on cellular adenosine triphosphate (ATP) contents. HH1023, the lot of hepatocytes with the highest CYP3A4 activity, was found to be the most sensitive to the cytotoxicity of all 4 hepatotoxic drugs, thereby suggesting that high CYP3A4 activity may be a risk factor. To further validate the relationship, a second study was performed with hepatocytes from 16 donors. In this study, the hepatocytes were quantified for CYP3A4 activity at the time of treatment. Results of the second study show confirm the correlation between with high CYP3A4 activity and sensitivity to hepatotoxic drugs. Our results with primary cultured hepatocytes from multiple donors support the hypothesis that elevated P450 activity may be a risk factor for drug-induced liver injuries. © 2015.

Doshi U.,In Vitro Admet Laboratories, Llc | Li A.P.,In Vitro Admet Laboratories, Llc
Journal of Biomolecular Screening | Year: 2011

The authors report here higher throughput screening (HTS) assays for the evaluation of CYP3A4 inhibition and CYP3A4 induction in human hepatocytes using a novel CYP3A4 substrate, luciferin IPA (LIPA). Using human recombinant CYP450 isoforms, LIPA was found to be metabolized extensively by CYP3A4 but not by CYP1A2, CYP2C9, CYP2C19, CYP2D6, or CYP2E1. In the 384-well plate CYP3A4 inhibition assay, the known inhibitors 1-aminobenzotriazole, erythromycin, ketoconazole, and verapamil were found to cause extensive (maximum inhibition of >80%), dose-dependent, statistically significant inhibition of LIPA metabolism. The non-CYP3A4 inhibitors diethyldithiocarbamate, quercetin, quinidine, sulfaphenazole, ticlopidine, and tranylcypromine were found to have substantially lower (maximum inhibition of <50%) or no apparent inhibitory effects in the HTS assay. In the 96-well plate induction assay, the CYP3A4 inducers rifampin, phenobarbital, carbamazepine, phenytoin, troglitazone, rosiglitazone, and pioglitazone yielded dose-dependent induction of LIPA metabolism, whereas the CYP1A2 inducers omeprazole and 3-methylcholanthrene did not display any induction in the CYP3A4 activity. The high sensitivity and specificity of the assays, the relative ease of execution, and reduced cost, time, and test material requirements suggest that the HTS assays may be applied routinely for screening a large number of chemicals in the drug discovery phase for CYP3A4 inhibitory and inducing potential. © 2011 Society for Laboratory Automation and Screening.

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.

In Vitro Admet Laboratories, Llc | Date: 2014-01-02

Cell growth media for growing cells for use in scientific research; Cells for scientific, laboratory or medical research. Culture fluids for cultivatinghepatocytes; Culture media for cultivatinghepatocytes. Bioreactor for cell culturing; Cell culture apparatus for laboratory use, namely, hepatocyte cultures.

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.

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