Modeling Therapeutic Antibody-Small Molecule Drug-Drug Interactions Using a Three-Dimensional Perfusable Human Liver Coculture Platform.

Traditional in vitro human liver cell culture models lose key hepatic functions such as metabolic activity during short-term culture. Advanced three-dimensional (3D) liver coculture platforms offer the potential for extended hepatocyte functionality and allow for the study of more complex biologic interactions, which can improve and refine human drug safety evaluations. Here, we use a perfusion flow 3D microreactor platform for the coculture of cryopreserved primary human hepatocytes and Kupffer cells to study the regulation of cytochrome P450 3A4 isoform (CYP3A4) activity by chronic interleukin 6 (IL-6)-mediated inflammation over 2 weeks.

Time-dependent cellular and transcriptional changes in the osteoblast lineage associated with sclerostin antibody treatment in ovariectomized rats.

Inhibition of sclerostin with sclerostin antibody (Scl-Ab) has been shown to stimulate bone formation, decrease bone resorption, and increase bone mass in both animals and humans. To obtain insight into the temporal cellular and transcriptional changes in the osteoblast (OB) lineage associated with long-term Scl-Ab treatment, stereological and transcriptional analyses of the OB lineage were performed on lumbar vertebrae from aged ovariectomized rats. Animals were administered Scl-Ab 3 or 50mg/kg/wk or vehicle (VEH) for up to 26weeks (d183), followed by a treatment-free period (TFP).

Utilization of human nuclear receptors as an early counter screen for off-target activity: a case study with a compendium of 615 known drugs.

Off-target effects of drugs on nuclear hormone receptors (NHRs) may result in adverse effects in multiple organs/physiological processes. Reliable assessments of the NHR activities for drug candidates are therefore crucial for drug development. However, the highly permissive structures of NHRs for vastly different ligands make it challenging to predict interactions by examining the chemical structures of the ligands.

Retinal Toxicity Induced by a Novel β-secretase Inhibitor in the Sprague-Dawley Rat.

β-Secretase 1 (BACE1) represents an attractive target for the treatment of Alzheimer's disease. In the course of development of a novel small molecule BACE1 inhibitor (AMG-8718), retinal thinning was observed in a 1-month toxicity study in the rat. To further understand the lesion, an investigational study was conducted whereby rats were treated daily with AMG-8718 for 1 month followed by a 2-month treatment-free phase.

A systematic assessment of mitochondrial function identified novel signatures for drug-induced mitochondrial disruption in cells.

Mitochondrial perturbation has been recognized as a contributing factor to various drug-induced organ toxicities. To address this issue, we developed a high-throughput flow cytometry-based mitochondrial signaling assay to systematically investigate mitochondrial/cellular parameters known to be directly impacted by mitochondrial dysfunction: mitochondrial membrane potential (MMP), mitochondrial reactive oxygen species (ROS), intracellular reduced glutathione (GSH) level, and cell viability. Modulation of these parameters by a training set of compounds, comprised of established mitochondrial poisons and 60 marketed drugs (30 nM to 1mM), was tested in HL-60 cells (a human pro-myelocytic leukemia cell line) cultured in either glucose-supplemented (GSM) or glucose-free (containing galactose/glutamine; GFM) RPMI-1640 media.

A multifactorial approach to hepatobiliary transporter assessment enables improved therapeutic compound development.

The bile salt export pump (BSEP) is expressed at the canalicular domain of hepatocytes, where it serves as the primary route of elimination for monovalent bile acids (BAs) into the bile canaliculi. The most compelling evidence linking dysfunction in BA transport with liver injury in humans is found with carriers of mutations that render BSEP nonfunctional. Based on mounting evidence, there appears to be a strong association between drug-induced BSEP interference and liver injury in humans; however, causality has not been established.

Raf inhibition causes extensive multiple tissue hyperplasia and urinary bladder neoplasia in the rat.

Seven novel and potent Raf small molecule kinase inhibitors (C1-7) were evaluated in seven-day oral repeat dose rat toxicity studies. All compounds tested induced hyperplasia in multiple tissues. Consistently affected was stratified squamous epithelium at a number of sites and transitional epithelium of urinary bladder and kidney.

A novel statistical algorithm for gene expression analysis helps differentiate pregnane X receptor-dependent and independent mechanisms of toxicity.

Genome-wide gene expression profiling has become standard for assessing potential liabilities as well as for elucidating mechanisms of toxicity of drug candidates under development. Analysis of microarray data is often challenging due to the lack of a statistical model that is amenable to biological variation in a small number of samples. Here we present a novel non-parametric algorithm that requires minimal assumptions about the data distribution.

The evolution of bioinformatics in toxicology: advancing toxicogenomics.

As one reflects back through the past 50 years of scientific research, a significant accomplishment was the advance into the genomic era. Basic research scientists have uncovered the genetic code and the foundation of the most fundamental building blocks for the molecular activity that supports biological structure and function. Accompanying these structural and functional discoveries is the advance of techniques and technologies to probe molecular events, in time, across environmental and chemical exposures, within individuals, and across species.

Interference with bile salt export pump function is a susceptibility factor for human liver injury in drug development.

The bile salt export pump (BSEP) is an efflux transporter, driving the elimination of endobiotic and xenobiotic substrates from hepatocytes into the bile. More specifically, it is responsible for the elimination of monovalent, conjugated bile salts, with little or no assistance from other apical transporters. Disruption of BSEP activity through genetic disorders is known to manifest in clinical liver injury such as progressive familial intrahepatic cholestasis type 2.

Application of genomics for identification of systemic toxicity triggers associated with VEGF-R inhibitors.

The key to the discovery of new pharmaceuticals is to develop molecules that interact with the intended target and minimize interaction with unintended molecular targets, therefore minimizing toxicity. This is aided by the use of various in vitro selectivity assays that are used to select agents most potent for the desired target. Typically, molecules from similar chemical series, with similar in vitro potencies, are expected to yield comparable in vivo pharmacological and toxicological profiles, predictive of target effects.

Current and future applications of toxicogenomics: Results summary of a survey from the HESI Genomics State of Science Subcommittee.

Background: In spite of the application of toxicogenomic (TGx) data to the field of toxicology for the past 10 years, the broad implementation and full impact of TGx for chemical and drug evaluation to improve decision making within organizations and by policy makers has not been achieved.

Objectives: The goal of the Health and Environmental Sciences Institute (HESI) Committee on the Application of Genomics to Mechanism-based Risk Assessment was to construct and summarize a multisector survey, addressing key issues and perspectives on the current and future practical uses and challenges of implementing TGx data to facilitate discussions for decision making within organizations and by policy makers.

Methods: An online survey to probe the current status and future challenges facing the field of TGx for drug and chemical evaluation in experimental and nonclinical models was taken by scientists and scientific decision/policy makers actively engaged in the field of TGx within industrial, academic, and regulatory sectors of the United States, Europe, and Japan.

Assessment of repeated microarray experiments using mixed tissue RNA reference samples.

Genome-scale gene expression technologies are increasingly being applied for biological research as a whole and toxicological screening in particular. In order to monitor data quality and process drift, we adopted the use of two rat-tissue mixtures (brain, liver, kidney, and testis) previously introduced as RNA reference samples. These samples were processed every time a microarray experiment was hybridized, thereby verifying the comparability of the resulting expression data for cross-study comparison.

Insight into redox-regulated gene networks in vascular cells.

To understand the complex nature of the atherogenic response initiated by oxidative stress in vascular smooth muscle cells (vSMCs), computational prediction methodology was employed to define putative gene-gene and gene-environment interactions in vSMCs subjected to oxidative chemical stress. Computational relationships were derived from the global gene expression profiles of murine cells challenged with a chemical pro-oxidant to cause oxidative stress or cells treated with anti-oxidant prior to oxidative injury. Target clones were chosen based on their biological relevance within the context of the atherogenic response and included lysyl oxidase, matrix metalloproteinase 2, insulin like growth factor binding protein 5, and lymphocyte antigen 6c.

Use of a mixed tissue RNA design for performance assessments on multiple microarray formats.

The comparability and reliability of data generated using microarray technology would be enhanced by use of a common set of standards that allow accuracy, reproducibility and dynamic range assessments on multiple formats. We designed and tested a complex biological reagent for performance measurements on three commercial oligonucleotide array formats that differ in probe design and signal measurement methodology. The reagent is a set of two mixtures with different proportions of RNA for each of four rat tissues (brain, liver, kidney and testes).

Prediction of nephrotoxicant action and identification of candidate toxicity-related biomarkers.

A vast majority of pharmacological compounds and their metabolites are excreted via the urine, and within the complex structure of the kidney,the proximal tubules are a main target site of nephrotoxic compounds. We used the model nephrotoxicants mercuric chloride, 2-bromoethylamine hydrobromide, hexachlorobutadiene, mitomycin, amphotericin, and puromycin to elucidate time- and dose-dependent global gene expression changes associated with proximal tubular toxicity. Male Sprague-Dawley rats were dosed via intraperitoneal injection once daily for mercuric chloride and amphotericin (up to 7 doses), while a single dose was given for all other compounds.

Integrating phenotypic and expression profiles to map arsenic-response networks.

Background: Arsenic is a nonmutagenic carcinogen affecting millions of people. The cellular impact of this metalloid in Saccharomyces cerevisiae was determined by profiling global gene expression and sensitivity phenotypes. These data were then mapped to a metabolic network composed of all known biochemical reactions in yeast, as well as the yeast network of 20,985 protein-protein/protein-DNA interactions.

Exploration of low-dose estrogen effects: identification of No Observed Transcriptional Effect Level (NOTEL).

Identifying a minimal dose capable of eliciting a biological response is a fundamental issue in a number of scientific fields, including: drug development, signal transduction research, and environmental toxicology. Frequently, proliferation, viability, and other assays based on the cellular response to a treatment are used to assess the threshold dose for minimal activity. Here we propose a novel approach for identifying the effects of low dose treatments and pinpointing the threshold dose.

Integration of clinical and gene expression endpoints to explore furan-mediated hepatotoxicity.

Molecular techniques, such as cDNA microarrays, are being used to aid in the elucidation of the mechanisms of toxicity of a variety of compounds. In this study, we evaluate the molecular effects of furan in the rat liver. Sprague-Dawley rats were exposed to 4 or 40 mg/kg furan for up to 14 days.

Short-chain fatty acids enhance nuclear receptor activity through mitogen-activated protein kinase activation and histone deacetylase inhibition.

In this study, we demonstrate that the pervasive xenobiotic methoxyacetic acid and the commonly prescribed anticonvulsant valproic acid, both short-chain fatty acids (SCFAs), dramatically increase cellular sensitivity to estrogens, progestins, and other nuclear hormone receptor ligands. These compounds do not mimic endogenous hormones but rather act to enhance the transcriptional efficacy of ligand activated nuclear hormone receptors by up to 8-fold in vitro and in vivo. Detailed characterization of their mode of action revealed that these SCFAs function as both activators of p42/p44 mitogen-activated protein kinase and as inhibitors of histone deacetylases at doses that parallel known exposure levels.

Histologically normal human mammary epithelia with silenced p16(INK4a) overexpress COX-2, promoting a premalignant program.

Breast tissue from healthy women contains variant mammary epithelial cells (vHMEC) exhibiting p16INK4a promoter hypermethylation both in vivo and in vitro. When continuously cultured, vHMEC acquire telomeric dysfunction and produce the types of chromosomal abnormalities seen in premalignant lesions of cancer. We find that late passage vHMEC express elevated prostaglandin cyclo-oxygenase 2 (COX-2), which contributes to increased prostaglandin synthesis, angiogenic activity, and invasive ability.

Identification of platform-independent gene expression markers of cisplatin nephrotoxicity.

Within the International Life Sciences Institute Committee on Genomics, a working group was formed to focus on the application of microarray technology to preclinical assessments of drug-induced nephrotoxicity. As part of this effort, Sprague-Dawley rats were treated with the nephrotoxicant cisplatin at doses of 0.3-5 mg/kg over a 4- to 144-hr time course.

Identification of putative gene based markers of renal toxicity.

This study, designed and conducted as part of the International Life Sciences Institute working group on the Application of Genomics and Proteomics, examined the changes in the expression profile of genes associated with the administration of three different nephrotoxicants--cisplatin, gentamicin, and puromycin--to assess the usefulness of microarrays in the understanding of mechanism(s) of nephrotoxicity. Male Sprague-Dawley rats were treated with daily doses of puromycin (5-20 mg/kg/day for 21 days), gentamicin (2-240 mg/kg/day for 7 days), or a single dose of cisplatin (0.1-5 mg/kg).