Identification of antibodies cross-reactive with woodchuck immune cells and activation of virus-specific and global cytotoxic T cell responses by anti-PD-1 and anti-PD-L1 in experimental chronic hepatitis B and persistent occult hepadnaviral infection.

Woodchuck (Marmota monax) infected with woodchuck hepatitis virus (WHV) is the most pathogenically compatible naturally occurring model of human hepatitis B virus (HBV) infection, chronic hepatitis B, and HBV-induced hepatocellular carcinoma. This system plays a crucial role in discovery and preclinical evaluation of anti-HBV therapies. Its utilization remains tempered by the relatively narrow range of validated immunologic and molecular tools. We evaluated commercial antibodies against immune cell phenotypic markers and T cell molecules for cross-reactivity with woodchuck antigenic equivalents. The confirmed antibodies against programed cell death protein-1 (PD-1) and its ligand (PD-L1) were examined for ex vivo ability to activate WHV-specific, global and bystander cytotoxic T cells (CTLs) in chronic hepatitis and asymptomatic infection persisting after self-resolved acute hepatitis. Examination of 65 antibodies led to identification or confirmation of 23 recognizing woodchuck T, regulatory T, B and natural killer cells, T cell-associated PD-1, PD-L1, CTLA-4 and TIM-3 molecules, CD25 and CD69 markers of T cell activation, and interferon gamma (IFNγ). Antibodies against woodchuck PD-1 and PD-L1 triggered in vitro highly individualized WHV-specific and global activation of CTLs in both chronic hepatitis and persistent occult infection. WHV-specific CTLs were more robustly augmented by anti-PD-1 than by anti-PD-L1 in chronic hepatitis, while global IFNγ-positive CTL response was significantly suppressed in chronic hepatitis compared to persistent occult infection. Anti-PD-1 and anti-PD-L1 also occasionally activated CTLs to specificities other than those tested suggesting their potency to trigger side effects. This was particularly apparent when T cells from chronic hepatitis were treated with anti-PD-L1. The current findings indicate that inhibition of the PD-1/PD-L1 pathway could reactivate virus-specific and global T cell responses in both chronic hepatitis and asymptomatic persistent infection. They suggest a mechanism of potential reactivation of clinically silent infection during anti-PD-1/PD-L1 treatment and indicate that this therapy may also subdue occult HBV infection.

Fitting tissue chips and microphysiological systems into the grand scheme of medicine, biology, pharmacology, and toxicology.

Microphysiological systems (MPS), which include engineered organoids (EOs), single organ/tissue chips (TCs), and multiple organs interconnected to create miniature in vitro models of human physiological systems, are rapidly becoming effective tools for drug development and the mechanistic understanding of tissue physiology and pathophysiology. The second MPS thematic issue of Experimental Biology and Medicine comprises 15 articles by scientists and engineers from the National Institutes of Health, the IQ Consortium, the Food and Drug Administration, and Environmental Protection Agency, an MPS company, and academia. Topics include the progress, challenges, and future of organs-on-chips, dissemination of TCs into Pharma, children's health protection, liver zonation, liver chips and their coupling to interconnected systems, gastrointestinal MPS, maturation of immature cardiomyocytes in a heart-on-a-chip, coculture of multiple cell types in a human skin construct, use of synthetic hydrogels to create EOs that form neural tissue models, the blood-brain barrier-on-a-chip, MPS models of coupled female reproductive organs, coupling MPS devices to create a body-on-a-chip, and the use of a microformulator to recapitulate endocrine circadian rhythms. While MPS hardware has been relatively stable since the last MPS thematic issue, there have been significant advances in cell sourcing, with increased reliance on human-induced pluripotent stem cells, and in characterization of the genetic and functional cell state in MPS bioreactors. There is growing appreciation of the need to minimize perfusate-to-cell-volume ratios and respect physiological scaling of coupled TCs. Questions asked by drug developers are followed by an analysis of the potential value, costs, and needs of Pharma. Of highest value and lowest switching costs may be the development of MPS disease models to aid in the discovery of disease mechanisms; novel compounds including probes, leads, and clinical candidates; and mechanism of action of drug candidates. Impact statement Microphysiological systems (MPS), which include engineered organoids and both individual and coupled organs-on-chips and tissue chips, are a rapidly growing topic of research that addresses the known limitations of conventional cellular monoculture on flat plastic - a well-perfected set of techniques that produces reliable, statistically significant results that may not adequately represent human biology and disease. As reviewed in this article and the others in this thematic issue, MPS research has made notable progress in the past three years in both cell sourcing and characterization. As the field matures, currently identified challenges are being addressed, and new ones are being recognized. Building upon investments by the Defense Advanced Research Projects Agency, National Institutes of Health, Food and Drug Administration, Defense Threat Reduction Agency, and Environmental Protection Agency of more than $200 million since 2012 and sizable corporate spending, academic and commercial players in the MPS community are demonstrating their ability to meet the translational challenges required to apply MPS technologies to accelerate drug development and advance toxicology.

Pharmacovigilance Assessment of Immune-Mediated Reactions Reported for Checkpoint Inhibitor Cancer Immunotherapies.

STUDY OBJECTIVE: Ipilimumab, pembrolizumab, and nivolumab are checkpoint inhibitors (CPIs) that activate T-cell-mediated immune response. CPI can provide durable benefits to some cancer patients with melanoma, renal cell cancer, non-small cell lung cancer, or a growing list of other cancers. However, CPI treatment is also associated with adverse immune-mediated reactions (IMRs) that can be life-threatening. This pharmacovigilance analysis aims to characterize IMR signals in relation to CPI treatment. DESIGN: Retrospective pharmacovigilance disproportionality analysis. DATA SOURCE: U.S. Food and Drug Administration Adverse Event Reporting System (FAERS). MEASUREMENTS AND MAIN RESULTS: Adverse event reports submitted to FAERS between 2011 and 2015 were analyzed. CPIs were identified by generic names, and IMRs were identified by MedDRA Preferred Terms. Empirical Bayes geometric means with corresponding 95% confidence intervals (EB05-EB95) were calculated as CPI-IMR association metrics. Signals were defined as metrics with EB05 ≥ 2.0. Overall, 1,018 IMR events were submitted for CPIs, corresponding to 76% for ipilimumab, 15% for nivolumab, and 9% for pembrolizumab. The period of data collection precluded data on the most recently approved CPI agents. IMRs consisted of 51% colitis, 16% endocrinopathies, 12% pneumonitis, 11% hepatitis, 4% infusion-related reactions, 3% nephritis, and 3% other IMRs. Colitis contributed to 63% and 41% of IMRs for ipilimumab and nivolumab, respectively. Pneumonitis and hepatitis contributed to a majority of IMRs for pembrolizumab, for which nephritis and infusion-related reactions were not reported. Signals of IMRs were detected for CPIs as a class (EB05 = 12.4) and individual agents: ipilimumab (EB05 = 13.2), nivolumab (EB05 = 15.0), and pembrolizumab (EB05 = 7.3). Colitis and pneumonitis had the strongest signals for CPIs (EB05 = 45.6 and EB05 = 17.6, respectively). Colitis was the strongest signal for ipilimumab (EB05 = 54.2), and pneumonitis was the strongest signal for nivolumab (EB05 = 48.0) and pembrolizumab (EB05 = 21.8). CONCLUSION: Cancer immunotherapy with CPIs is associated with a multitude of IMRs, especially colitis and pneumonitis. Individual CPIs had variable IMR signals, and pharmacoepidemiologic studies are required to evaluate the identified signals.

The Rat microRNA body atlas; Evaluation of the microRNA content of rat organs through deep sequencing and characterization of pancreas enriched miRNAs as biomarkers of pancreatic toxicity in the rat and dog.

BACKGROUND: MicroRNAs (miRNA) are ~19-25 nucleotide long RNA molecules that fine tune gene expression through the inhibition of translation or degradation of the mRNA through incorporation into the RNA induced silencing complex (RISC). MicroRNAs are stable in the serum and plasma, are detectable in a wide variety of body fluids, are conserved across veterinary species and humans and are expressed in a tissue specific manner. They can be detected at low concentrations in circulation in animals and humans, generating interest in the utilization of miRNAs as serum and/or plasma based biomarkers of tissue injury. MicroRNA tissue profiling in rodents has been published, but sample an insufficient number of organs of toxicologic interest using microarray or qPCR technologies for miRNA detection. Here we impart an improved rat microRNA body atlas consisting of 21 and 23 tissues of toxicologic interest from male and female Sprague Dawley rats respectively, using Illumina miRNA sequencing. Several of the authors created a dog miRNA body atlas and we collaborated to test miRNAs conserved in rat and dog pancreas in caerulein toxicity studies utilizing both species. RESULTS: A rich data set is presented that more robustly defines the tissue specificity and enrichment profiles of previously published and undiscovered rat miRNAs. We generated 1,927 sequences that mapped to mature miRNAs in rat, mouse and human from miRBase and discovered an additional 1,162 rat miRNAs as compared to the current number of rat miRNAs in miRBase version 21. Tissue specific and enriched miRNAs were identified and a subset of these miRNAs were validated by qPCR for tissue specificity or enrichment. As an example of the power of this approach, we have conducted rat and dog pancreas toxicity studies and examined the levels of some tissue specific and enriched miRNAs conserved between rat and dog in the serum of each species. The studies demonstrate that conserved tissue specific/enriched miRs-216a-5p, 375-3p, 148a-3p, 216b-5p and 141-3p are candidate biomarkers of pancreatic injury in the rat and dog. CONCLUSIONS: A microRNA body atlas for rat and dog was useful in identifying new candidate miRNA biomarkers of organ toxicity in 2 toxicologically relevant species.

Evaluation of the Relative Performance of Drug-Induced Skeletal Muscle Injury Biomarkers in Rats.

Novel skeletal muscle (SKM) injury biomarkers that have recently been identified may outperform or add value to the conventional SKM injury biomarkers aspartate transaminase (AST) and creatine kinase (CK). The relative performance of these novel biomarkers of SKM injury including skeletal troponin I (sTnI), myosin light chain 3 (Myl3), CK M Isoform (Ckm), and fatty acid binding protein 3 (Fabp3) was assessed in 34 rat studies including both SKM toxicants and compounds with toxicities in tissues other than SKM. sTnI, Myl3, Ckm, and Fabp3 all outperformed CK or AST and/or added value for the diagnosis of drug-induced SKM injury (ie, myocyte degeneration/necrosis). In addition, when used in conjunction with CK and AST, sTnI, Myl3, CKm, and Fabp3 individually and collectively improved diagnostic sensitivity and specificity, as well as diagnostic certainty, for SKM injury and responded in a sensitive manner to low levels of SKM degeneration/necrosis in rats. These findings support the proposal that sTnI, Myl3, Ckm, and Fabp3 are suitable for voluntary use, in conjunction with CK and AST, in regulatory safety studies in rats to monitor drug-induced SKM injury and the potential translational use of these exploratory biomarkers in early clinical trials to ensure patient safety.

Natriuretic Peptides as Cardiovascular Safety Biomarkers in Rats: Comparison With Blood Pressure, Heart Rate, and Heart Weight.

Cardiovascular (CV) toxicity is an important cause of failure during drug development. Blood-based biomarkers can be used to detect CV toxicity during preclinical development and prioritize compounds at lower risk of causing such toxicities. Evidence of myocardial degeneration can be detected by measuring concentrations of biomarkers such as cardiac troponin I and creatine kinase in blood; however, detection of functional changes in the CV system, such as blood pressure, generally requires studies in animals with surgically implanted pressure transducers. This is a significant limitation because sustained changes in blood pressure are often accompanied by changes in heart rate and together can lead to cardiac hypertrophy and myocardial degeneration in animals, and major adverse cardiovascular events (MACE) in humans. Increased concentrations of NPs in blood correlate with higher risk of cardiac mortality, all-cause mortality, and MACE in humans. Their utility as biomarkers of CV function and toxicity in rodents was investigated by exploring the relationships between plasma concentrations of NTproANP and NTproBNP, blood pressure, heart rate, and heart weight in Sprague Dawley rats administered compounds that caused hypotension or hypertension, including nifedipine, fluprostenol, minoxidil, L-NAME, L-thyroxine, or sunitinib for 1-2 weeks. Changes in NTproANP and/or NTproBNP concentrations were inversely correlated with changes in blood pressure. NTproANP and NTproBNP concentrations were inconsistently correlated with relative heart weights. In addition, increased heart rate was associated with increased heart weights. These studies support the use of natriuretic peptides and heart rate to detect changes in blood pressure and cardiac hypertrophy in short-duration rat studies.

Evaluation of Efficacy and Safety of the Glucagon Receptor Antagonist LY2409021 in Patients With Type 2 Diabetes: 12- and 24-Week Phase 2 Studies.

OBJECTIVE: Type 2 diabetes pathophysiology is characterized by dysregulated glucagon secretion. LY2409021, a potent, selective small-molecule glucagon receptor antagonist that lowers glucose was evaluated for efficacy and safety in patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: The efficacy (HbA1c and glucose) and safety (serum aminotransferase) of once-daily oral administration of LY2409021 was assessed in two double-blind studies. Phase 2a study patients were randomized to 10, 30, or 60 mg of LY2409021 or placebo for 12 weeks. Phase 2b study patients were randomized to 2.5, 10, or 20 mg LY2409021 or placebo for 24 weeks. RESULTS: LY2409021 produced reductions in HbA1c that were significantly different from placebo over both 12 and 24 weeks. After 12 weeks, least squares (LS) mean change from baseline in HbA1c was -0.83% (10 mg), -0.65% (30 mg), and -0.66% (60 mg) (all P < 0.05) vs. placebo, 0.11%. After 24 weeks, LS mean change from baseline in HbA1c was -0.45% (2.5 mg), -0.78% (10 mg, P < 0.05), -0.92% (20 mg, P < 0.05), and -0.15% with placebo. Increases in serum aminotransferase, fasting glucagon, and total fasting glucagon-like peptide-1 (GLP-1) were observed; levels returned to baseline after drug washout. Fasting glucose was also lowered with LY2409021 at doses associated with only modest increases in aminotransferases (mean increase in alanine aminotransferase [ALT] ≤10 units/L). The incidence of hypoglycemia in the LY2409021 groups was not statistically different from placebo. CONCLUSIONS: In patients with type 2 diabetes, glucagon receptor antagonist treatment significantly lowered HbA1c and glucose levels with good overall tolerability and a low risk for hypoglycemia. Modest, reversible increases in serum aminotransferases were observed.

Initial biological qualification of SBDP-145 as a biomarker of compound-induced neurodegeneration in the rat.

Detection of compound-related neurodegeneration is currently limited to brain histopathology in veterinary species and functional measurements such as electroencephalography and observation of clinical signs in patients. The objective of these studies was to investigate whether concentrations of spectrin breakdown product 145 (SBDP-145) in cerebrospinal fluid (CSF) correlate with the severity of neurodegeneration in rats administered neurotoxic agents, as part of a longer term objective of developing in vivo biomarkers of neurotoxicity for use in non-clinical and clinical safety studies. Non-erythroid alpha-II spectrin is a cytoskeletal protein cleaved by the protease calpain when this enzyme is activated by dysregulation of calcium in injured cells. Calcium dysregulation is also associated with some toxicological responses in animals, and may be sufficient to activate neuronal calpain and produce SBDPs that can be released into CSF. Neurotoxicants (kainic acid, 2-chloropropionic acid, bromethalin, and pentylenetetrazole) known to affect different portions of the brain were administered to rats in dose-response and time-course studies in which neurodegeneration was measured by histopathology and SBDP-145 concentrations in CSF were measured by ELISA. We consistently observed >3-fold increases in SBDP-145 concentration in rats with minimal to slight neurodegenerative lesions, and 20 to 150-fold increases in animals with more severe lesions. In contrast, compounds that caused non-degenerative changes in central nervous system (CNS) did not increase SBDP-145 in CSF. These data support expanded use of SBDP-145 as a biomarker for monitoring compound-induced neurodegeneration in pre-clinical studies, and support the investigation of clinical applications of this biomarker to promote safe dosing of patients with compounds that have potential to cause neurodegeneration.

Biomarkers of exocrine pancreatic injury in 2 rat acute pancreatitis models.

UNLABELLED: Consistent, sensitive biomarkers of exocrine pancreatic injury (EPIJ) in animal models and humans have historically represented a poorly met need for investigators and clinicians. EXPERIMENTAL DESIGN: Sprague-Dawley CD/International Genetic Standard system (IGS) rats were administered cerulein or cyanohydroxybutene (CHB) to induce EPIJ. Serum samples were taken at time points between 1- and 168-hr postinjection (PI), and rats were sacrificed between 24- and 168-hr PI. METHOD: We investigated a series of serum-based biomarkers including amylase, lipase, pancreas-enriched microRNAs (miRs) and inflammation biomarkers compared with concurrent hematology and pancreatic histology. RESULTS AND CONCLUSION: Microscopic EPIJ was not associated with consistent changes in hematology or inflammation biomarkers. Increased severity scores for EPIJ correlated with increased amylase and lipase values, although severity of EPIJ did not always correlate with the magnitude of enzyme increases. Microscopic EPIJ was most severe at 24 to 48 hr; increases in miR-216a (32-fold) and miR-375 (23-fold) were present at 24 hr and, along with enzymes, were normalized by 48 hr in the cerulein study. MiRs-216a and 375 were increased by ∼800- and 500-fold, respectively, at 24 hr while miR-375 remained elevated until 72 hr in the CHB study. Impact statement: Pancreas-enriched miRs hold promise as novel serum-based biomarkers for EPIJ.

A novel high-sensitivity electrochemiluminescence (ECL) sandwich immunoassay for the specific quantitative measurement of plasma glucagon.

OBJECTIVES: To develop a novel, dual-monoclonal sandwich immunoassay with superior sensitivity that provides a rapid and convenient method for measuring glucagon. Glucagon is a 29-amino acid polypeptide hormone produced in the pancreas by the α-cells of the islets of Langerhans. Working in concert with insulin, glucagon is involved in regulating circulating glucose concentrations. DESIGN AND METHODS: The immunoassay utilizes Meso Scale Discovery (MSD) electrochemiluminescence (ECL) technology and two affinity-optimized monoclonal antibodies. A series of experiments was performed to determine the linear range of the assay and to evaluate sensitivity, accuracy, recovery, precision, and linearity. RESULTS: The sandwich assay was specific for glucagon and did not recognize the closely related peptide oxyntomodulin or other incretin peptides. The assay demonstrated excellent recovery, precision, and linearity, and a broad dynamic range of 0.14 pmol/L to 1950 pmol/L. In addition, assay results were highly correlated with those obtained using a previously described competitive RIA employing polyclonal antiserum. CONCLUSION: The use of affinity-optimized monoclonal antibodies in a sandwich immunoassay format provides a robust, sensitive, and convenient method for measuring concentrations of glucagon that is highly sensitive and specific. This immunoassay should help to improve our understanding of the role of glucagon in the regulation of glucose metabolism.

Relating molecular properties and in vitro assay results to in vivo drug disposition and toxicity outcomes.

A primary goal of lead optimization is to identify compounds with improved absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties. A number of reports have linked computed molecular properties to desirable in vivo ADMET outcomes, but a significant limitation of these analyses is the failure to control statistically for possible covariates. We examine the relationship between molecular properties and in vitro surrogate assays vs in vivo properties within 173 chemical series from a database of 3773 compounds with rodent pharmacokinetic and toxicology data. This approach identifies the following pairs of surrogates as most predictive among those examined: rat primary hepatocyte (RPH) cytolethality/volume of distribution (V(d)) for in vivo toxicology outcomes, scaled microsome metabolism/calculated logP for in vivo unbound clearance, and calculated logD/kinetic aqueous solubility for thermodynamic solubility. The impact of common functional group substitutions is examined and provides insights for compound design.

Detection of left ventricular hypertrophy in rats administered a peroxisome proliferator-activated receptor alpha/gamma dual agonist using natriuretic peptides and imaging.

Chronic treatment with suprapharmacologic doses of peroxisome proliferator-activated receptor (PPAR) agonists has a known potential for causing left ventricular hypertrophy (LVH). The mechanism by which LVH develops is not well understood nor are biomarkers of it well characterized. Natriuretic peptides are important regulators of cardiac growth, blood volume, and arterial pressure and may be useful biomarkers of LVH and hemodynamic changes that precede it. We measured amino-terminal pro-atrial natriuretic peptide (NTproANP), amino-terminal pro-brain natriuretic peptide (NTproBNP), and cardiac troponin I (cTnI) concentrations in serum and plasma, as well as transcripts in left ventricular heart tissue for atrial natriuretic peptide precursor (Nppa), brain natriuretic peptide precursor (Nppb), and myosin heavy chain-beta (Myh7) as potential biomarkers of LVH induced by a PPARalpha/gamma dual agonist in Sprague-Dawley rats. We used magnetic resonance imaging, echocardiography, and hemodynamics to identify structural and functional cardiovascular changes related to the biomarkers. Heart-to-brain weight ratios (HW:BrW) were correlated with NTproANP, NTproBNP, and cTnI concentrations in serum as well as fold change in expression of Nppa and Nppb. LVH was characterized by increased left ventricular wall thickness and inner diameter, increased cardiac output, decreased arterial blood pressure, and increased heart rate. In these studies, each end point contributed to the early detection of LVH, the ability to monitor its progression, and demonstrated the ability of NTproANP concentration in serum to predict LVH and hemodynamic changes.

Electrochemiluminescent immunoassay for rat skeletal troponin I (Tnni2) in serum.

INTRODUCTION: The identification of xenobiotic-induced skeletal muscle toxicities through the detection of biomarkers in nonclinical studies can be useful early in the drug discovery process to aid in candidate drug decisions. Skeletal muscle troponin I (sTnI) has been identified as a potential marker of skeletal muscle injury in humans and animals. When skeletal muscle tissue is injured, sTnI is released into circulation. METHODS: Due to the nature of the troponin subunits to form intermolecular complexes and to oxidize under various environmental conditions, the optimal assay required the use of a combination of chelating and reducing agents in the sample preparation. It also required the selection of capture and detection antibodies with specificity to the reduced sTnI monomeric subunit and includes a capture antibody specific for sTnI Type 2 (Tnni2), which is associated with Type 2 "fast twitch" muscle fibers. RESULTS: We have developed a sensitive and specific assay to detect the concentration of rat sTnI in serum using an electrochemiluminescent (ECL) immunoassay platform with a sensitivity of 2.4 ng/ml and with minimal cross-reactivity with rat cardiac TnI (Tnni3). DISCUSSION: The use of additives and the wide dynamic range of the ECL platform resulted in an accurate and consistent ECL immunoassay that was able to specifically detect sTnI (Tnni2) in rat serum. This method can be applied to safety assessment in early drug development.

Qualification of cardiac troponin I concentration in mouse serum using isoproterenol and implementation in pharmacology studies to accelerate drug development.

Cardiac troponin I is a useful biomarker of myocardial injury, but its use in mice and application to early drug discovery are not well described. The authors investigated the relationship between cTnI concentration in serum and histologic lesions in heart tissue from mice treated with isoproterenol (ISO). Cardiac TnI concentrations in serum increased in a dose-dependant manner and remained increased twenty-four to forty-eight hours after a single administration of isoproterenol. Increased cTnI concentration was of greater magnitude and longer duration than increased fatty acid binding protein 3 concentration, aspartate aminotransferase activity, and creatine kinase activity in serum. Isoproterenol-induced increases in cTnI concentrations were both greater and more sustained in BALB/c than in CD1 mice and correlated with incidence and severity of lesions observed in heart sections from both strains. In drug development studies in BALB/c mice with novel kinase inhibitors, cTnI concentration was a reliable stand-alone biomarker of cardiac injury and was used in combination with measurements of in vivo target inhibition to demonstrate an off-target contribution to cardiotoxicity. Additional attributes, including low cost and rapid turnaround time, made cTnI concentration in serum invaluable for detecting cardiotoxicity, exploring structure-activity relationships, and prioritizing development of compounds with improved safety profiles early in drug discovery.

Fabp3 as a biomarker of skeletal muscle toxicity in the rat: comparison with conventional biomarkers.

Fatty acid binding protein 3 (Fabp3) has been used as a serological biomarker of cardiac injury, but its utility as a preclinical biomarker of injury to skeletal muscle is not well described. Fabp3 concentrations were determined for tissues from Sprague-Dawley rats and found to occur at highest concentrations in cardiac muscle and in skeletal muscles containing an abundance of type I fibers, such as the soleus muscle. Soleus is also a primary site of skeletal muscle (SKM) injury caused by lipid-lowering peroxisome proliferator-activated receptor alpha (PPAR-alpha) agonists. In rats administered repeat doses of a PPAR-alpha agonist, the kinetics and amplitude of plasma concentrations of Fabp3 were consistent with plasma compound concentrations and histopathology findings of swollen, hyalinized, and fragmented muscle fibers with macrophage infiltration. Immunohistochemical detection of Fabp3 revealed focal depletion of Fabp3 protein from injured SKM fibers which is consistent with increased serum Fabp3 concentrations in treated rats. We then assessed the predictivity of serological Fabp3 for SKM necrosis in short duration toxicology studies. Rats were treated with various doses of 27 different compounds, and the predictivity of serological biomarkers was assessed relative to histology in individual rats and in treatment groups. Under these study conditions, Fabp3 was the most useful individual biomarker based on concordance, sensitivity, positive and negative predictive values, and false negative rate. In addition, the combination of Fabp3 and aspartate aminotransferase (AST) had greater diagnostic value than the conventional combination of creatine kinase-MM isoenzyme (CK) and AST.

Strategic use of immunoprecipitation and LC/MS/MS for trace-level protein quantification: myosin light chain 1, a biomarker of cardiac necrosis.

Myosin light chain 1 (Myl3) is a 23-kDa isoform of one of the subunits of myosin, a protein involved in muscle contraction. Myl3 is presently being studied as a biomarker of cardiac necrosis to predict drug-induced cardiotoxicity, and in the work presented here, an LC/MS/MS assay was developed and validated to measure Myl3 in rat serum. The key steps in this approach involved immunoaffinity purification of Myl3 from serum followed by on-bead digestion with trypsin to release a surrogate peptide. This tryptic peptide was quantified using a synthetic peptide standard and a corresponding stable isotope-labeled internal standard, and the results were stoichiometrically converted to Myl3 serum concentrations. Myl3 concentrations were corrected for peptide recovery following immunoprecipitation and digestion (85%) and showed excellent agreement with synthetic peptide standards. Both the synthetic peptide and His-Myl3 protein were used to evaluate assay accuracy (% RE) and precision (% CV), which were measured on each of 3 days. The synthetic peptide was evaluated over the range of 0.073-7.16 nM, while Myl3 protein QC samples prepared in rat serum were evaluated over the range of 0.13-6.62 nM. To prepare control matrix, endogenous Myl3 was immunodepleted from pooled rat serum. Peptide interday accuracy and precision did not exceed 7.6 and 11.1%, and Myl3 interday accuracy and precision did not exceed 12.9 and 13.2%, respectively. Data are presented from the application of this assay to establish a time course in which rats demonstrated a marked increase in Myl3 serum concentrations following administration of isoproterenol, a beta-adrenergic receptor agonist known to induce cardiac injury. This assay is an example of a larger effort in our laboratory to use LC/MS/MS in conjunction with immunoaffinity techniques to evaluate candidate biomarkers of target organ toxicity and to expedite the development of biomarker assays for drug development.

Quantification of heart fatty acid binding protein as a biomarker for drug-induced cardiac and musculoskeletal necroses.

Heart fatty acid binding protein (Fabp3) is a cytosolic protein expressed primarily in heart, and to a lesser extent in skeletal muscle, brain, and kidney. During myocardial injury, the Fabp3 level in serum is elevated rapidly, making it an ideal early marker for myocardial infarction. In this study, an MS-based selected reaction monitoring method (LC-SRM) was developed for quantifying Fabp3 in rat serum. Fabp3 was enriched first through an immobilized antibody, and the protein was digested on beads directly. A marker peptide of Fabp3 was quantified using LC-SRM with a stable isotope-labeled peptide standard. For six quality control samples with Fabp3 ranging from 0.256 to 25 ng, the average recovery following the procedure was about 73%, and the precision (%CV) between replicates was less than 7%. The Fabp3 concentrations in rat serum peaked 1 h after isoproterenol treatment, and returned to baseline levels 24 h after the dose. Elevated Fabp3 levels were also detected in rats administered with a PPAR α/δ agonist, which has shown to cause skeletal muscle necrosis. Fabp3 can be used as a biomarker for both cardiac and skeletal necroses. The cross-validation of the LC-SRM method with an existing ELISA method is described.

Relationships between genomic, cell cycle, and mutagenic responses of TK6 cells exposed to DNA damaging chemicals.

Genotoxic stress causes a variety of cellular and molecular responses in mammalian cells, including cell cycle arrest, DNA repair, and apoptosis. These responses result from the interplay between the genotoxic events themselves, and the biological context in which they occur. To better understand this interplay, we investigated cytotoxicty, mutagenesis, cell cycle profile, and global gene expression in the human TK6 lymphoblastoid cell line exposed to six genotoxicants. The six compounds have broad structural diversity and cause genotoxic stress by many different mechanisms, including covalent modification (methyl methanesulfonate, mitomycin C), reactive oxygen species (hydrogen peroxide, bleomycin), and topoisomerase II inhibition (etoposide and doxorubicin). Cell cycle analysis was performed 4 and 20 h following a 4 h chemical exposure. Cells exposed to all compounds experienced S-phase arrest at the 8h time point, but by 24 h had markedly different cell cycle responses. Cells exposed to compounds that cause covalent modification had a strong G2/M arrest at 24 h. These cells also had a robust (>25-fold) increase in mutant frequency, and had a moderate but sustained p53 response at 4, 8, and 24h, detectable as approximately 2-5-fold increases in transcript levels for p21WAF1/CIP1, GADD45alpha, BTG2, and cyclin G1. In contrast, cells exposed to the reactive oxygen compounds had little or no G2/M arrest at 24 h and no increase in mutant frequency. In addition, these compounds caused a strong but transient induction of the p53 pathway, detectable as 15-25-fold increases in p21WAF1/CIP1 transcription at 4 h that decreased dramatically by 8h and was near control levels at 24 h. Thus, the mutagenic effect of compounds was consistent with G2/M arrest and sustained kinetics of p53 pathway activation. Global gene expression data were also consistent with the mutagenesis data. Activation of genes associated with cell cycle arrest, the p53 and TNF-related pathways, and chemokines and chemokine receptors, were particularly evident for the reactive oxygen compounds. In contrast, the most mutagenic compounds caused fewer and less robust changes in global gene expression. There was therefore an inverse relationship between global gene expression and mutagenic potency.