Myrtle A. Davis

Renal biomarker qualification submission: a dialog between the FDA-EMEA and Predictive Safety Testing Consortium

The first formal qualification of safety biomarkers for regulatory decision making marks a milestone in the application of biomarkers to drug development. Following submission of drug toxicity studies and analyses of biomarker performance to the Food and Drug Administration (FDA) and European Medicines Agency (EMEA) by the Predictive Safety Testing Consortiums (PSTC) Nephrotoxicity Working Group, seven renal safety biomarkers have been qualified for limited use in nonclinical and clinical drug development to help guide safety assessments. This was a pilot process, and the experience gained will both facilitate better understanding of how the qualification process will probably evolve and clarify the minimal requirements necessary to evaluate the performance of biomarkers of organ injury within specific contexts.

Hepatic Gene Expression Changes in Mice Associated with Prolonged Sublethal Microcystin Exposure

Microcystin-LR (MCLR) is an acute hepatotoxicant and suspected carcinogen. Previous chronic studies have individually described hepatic morphologic changes, or alterations in the cytoskeleton, cell signaling or redox pathways. The objective of this study was to characterize chronic effects of MCLR in wild-type mice utilizing gene array analysis, morphology, and plasma chemistries. MCLR was given daily for up to 28 days. RNA from the 28-day study was hybridized onto mouse genechip arrays. RNA from 4 hours, 24 hours, 4 days, 1 day, and 28 days for selected genes was processed for quantitative-PCR. Increases in plasma hepatic enzyme activities and decreases in total protein, albumin and glucose concentrations were identified in MCLR-treated groups at 14 and 28 days. Histologically, marked hepatokaryomegaly was identified in the 14-day MCLR group with the addition of giant cells at 28 days. Major gene transcript changes were identified in the actin organization, cell cycle, apoptotic, cellular redox, cell signaling, albumin metabolism, and glucose homeostasis pathways, and the organic anion transport polypeptide system. Using toxicogenomics, we have identified key molecular pathways involved in chronic sublethal MCLR exposure in wild-type mice, genes participating in those critical pathways and related them to cellular and morphologic alterations seen in this and other studies.

Review Article: Apoptosis in the Kidney

Apoptosis is a highly regulated mechanism of cell death. Although apoptosis has a functional role in normal development and tissue homeostasis, aberrant triggering of the process by toxicants may lead to abnormal function or disease. Low level exposures to toxicants that induce apoptosis in kidney may therefore create a critical disturbance in kidney homeostasis, contributing to renal neoplasia or renal disease. In this report, we review the involvement of apoptosis in normal kidney development and in renal disease and discuss some of the toxicants and molecular factors involved in regulation of the process in renal cells.

Characterization of a Renal Epithelial Cell Model of Apoptosis Using Okadaic Acid and the NRK-52E Cell Line

Apoptotic cell death plays an important role in the pathogenesis of renal tubular epithelial damage and repair following tubular injury. Presently, the cellular factors involved in regulating apoptotic pathways in the kidney are unknown. To address the possibility that protein phosphorylation may regulate apoptosis in kidney cells, okadaic acid (OKA), a specific inhibitor of protein phosphatases 1A and 2A, was tested for its morphologic and biochemical effects on normal rat kidney epithelial cells (NRK-52E) in culture. As revealed by the DNA-specific stain DAPI, nuclei of cells treated with 1.0 μM okadaic acid contained irregular clumps of dense chromatin. Additional morphologic alterations typical of apoptosis were apparent within 2 hr after treatment with 1.0 μM OKA, including marked cellular rounding, cytoplasmic condensation, and cytoplasmic blebs. Ultrastructurally, 1.0 μM OKA caused cytoplasmic bleb formation, cellular fragmentation, condensation of heterochromatin into clumps, and segregation of nucleoli. At this stage, the cytoplasmic fragments and blebs contained many normal mitochondria. The attached, rounded cells also effectively excluded propidium iodide, demonstrating maintenance of membrane integrity despite pronounced morphologic alterations. A 2-fold increase in intracellular free Ca2+ was apparent 90 min after treatment with 1.0 μM okadaic acid. Transverse alternating field electrophoresis revealed the appearance of large DNA fragments of approximately 300-kbp. The appearance of these 300-kbp fragments correlated temporally with the observed elevation in intracellular calcium and the onset of morphologic alterations. However, preloading cells with EGTA-AM, an intracellular calcium chelator, obliterated the calcium elevation and had no effect on OKA-induced morphology, DNA fragmentation, or cell death. Detectable internucleosomal fragmentation occurred much later than the onset of morphologic changes (24-hr treatment time) and did not correlate with elevations in cellular calcium. These studies support the hypothesis that during apoptosis, chromatin condensation reflects chromatin cleavage at nuclease-sensitive sites between hexameric rosettes. These results also suggest that morphologic and nuclear alterations in the pathway of OKA-induced apoptosis occur independent of observed increases in intracellular calcium.

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.

Chronic microcystin exposure induces hepatocyte proliferation with increased expression of mitotic and cyclin-associated genes in P53-deficient mice.

Homozygous p53 deficient knockout mice were used to assess the role of p53 in tumor promotion by the protein phosphatase inhibitor and hepatic tumor promoter microcystin-LR (MCLR). More than 50% of human cancers bear mutations in the p53 gene, and in particular, p53 tumor suppressor gene mutations have been shown to play a major role in hepatocarcinogenesis. Trp53 homozygous (inactivated p53) and age-matched wild-type control mice were assigned to vehicle or MCLR-treated groups. MCLR or saline was administered daily for up to 28 days. RNA from the 28-day study was hybridized onto Mouse Genome GeneChip arrays. Selected RNA from 28 days and earlier time points was also processed for quantitative polymerase chain reaction (PCR). Livers from the 28-day, Trp53-deficient, MCLR group displayed greater hyperplastic and dysplastic changes morphologically and increases in Ki-67 and phosphohistone H3 (mitotic marker) immunoreactivity. Gene-expression analysis revealed significant increases in expression of cell-cycle regulation and cellular proliferation genes in the MCLR-treated, p53-deficient mutant mice compared to controls. These data suggest that regulation of the cell cycle by p53 is important in preventing the proliferative response associated with chronic, sublethal microcystin exposure, and therefore, conclude that p53 plays an important role in MCLR-induced tumor promotion.

Examining the Protective Role of ErbB2 Modulation in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are being used as an in vitro model system in cardiac biology and in drug discovery (e.g., cardiotoxicity testing). Qualification of these cells for use in mechanistic investigations will require detailed evaluations of cardiomyocyte signaling pathways and cellular responses. ErbB signaling and the ligand neuregulin play critical roles in survival and functional integrity of cardiac myocytes. As such, we sought to characterize the expression and activity of the ErbB family of receptors. Antibody microarray analysis performed on cell lysates derived from maturing hiPSC-CMs detected expression of ∼570 signaling proteins. EGFR/ErbB1, HER2/ErbB2, and ErbB4, but not ErbB3 receptors, of the epidermal growth factor receptor family were confirmed by Western blot. Activation of ErbB signaling by neuregulin-1β (NRG, a natural ligand for ErbB4) and its modulation by trastuzumab (a monoclonal anti-ErbB2 antibody) and lapatinib (a small molecule ErbB2 tyrosine kinase inhibitor) were evaluated through assessing phosphorylation of AKT and Erk1/2, two major downstream kinases of ErbB signaling, using nanofluidic proteomic immunoassay. Downregulation of ErbB2 expression by siRNA silencing attenuated NRG-induced AKT and Erk1/2 phosphorylation. Activation of ErbB signaling with NRG, or inhibition with trastuzumab, alleviated or aggravated doxorubicin-induced cardiomyocyte damage, respectively, as assessed by a real-time cellular impedance analysis and ATP measurement. Collectively, these results support the expanded use of hiPSC-CMs to examine mechanisms of cardiotoxicity and support the value of using these cells in early assessments of cardiotoxicity or efficacy.

The new revolution in toxicology: the good, the bad, and the ugly.

In 2007, the United States National Academy of Sciences issued a report entitled Toxicity Testing in the 21st Century: A Vision and a Strategy. The report reviewed the state of the science and outlined a strategy for the future of toxicity testing. One of the more significant components of the vision established by the report was an emphasis on toxicity testing in human rather than animal systems. In the context of drug development, it is critical that the tools used to accomplish this strategy are maximally capable of evaluating human risk. Since 2007, many advances toward implementation of this vision have been achieved, particularly with regard to safety assessment of new chemical entities intended for pharmaceutical use.

Identification and Elucidation of the Biology of Adverse Events: The Challenges of Safety Assessment and Translational Medicine

There has been an explosion of technology-enabled scientific insight into the basic biology of the causes of adverse events. This has been driven, in part, by the development of the various “omics” tools (e.g., genomics, proteomics, and metabolomics) and associated bioinformatics platforms. Meanwhile, for decades, changes in preclinical testing protocols and guidelines have been limited. Preclinical safety testing currently relies heavily on the use of outdated animal models. Application of systems biology methods to evaluation of toxicities in oncology treatments can accelerate the introduction of safe, effective drugs. Systems biology adds insights regarding the causes and mechanisms of adverse effects, provides important and actionable information to help understand the risks and benefits to humans, focuses testing on methods that add value to the safety testing process, and leads to modifications of chemical entities to reduce liabilities during development. Leveraging emerging technologies, such as genomics and proteomics, may make preclinical safety testing more efficient and accurate and lead to better safety decisions. The development of a U.S. Food and Drug Administration guidance document on the use of systems biology in clinical testing would greatly benefit the development of drugs for oncology by communicating the potential application of specific methodologies, providing a framework for qualification and application of systems biology outcomes, and providing insight into the challenges and limitations of systems biology in the regulatory decision-making process. Clin Cancer Res; 17(21); 6641–5. ©2011 AACR.

In Vitro Exposure of Precision-Cut Lung Slices to 2-(4-Amino-3-Methylphenyl)-5-Fluorobenzothiazole Lysylamide Dihydrochloride (NSC 710305, Phortress) Increases Inflammatory Cytokine Content and Tissue Damage

The anticancer drug (2-[4-amino-3-methylphenyl]-5-fluorobenzothiazole lysylamide dihydrochloride) (NSC 710305, Phortress) is a metabolically activated prodrug that causes DNA adduct formation and subsequent toxicity. Preclinically, it was found that hepatic, bone marrow, and pulmonary toxicity presented challenges to developing this drug. An ex vivo precision-cut lung slice (PCLS) model was used to search for concentration dependent effects of NSC 710305 (10, 25, 50, and 100 µM) on cytokine content, protein content, and immuno/histological endpoints. Preparation and culture of PCLS caused an initial spike in proinflammatory cytokine expression and therefore treatment with NSC 710305 was delayed until 48 h after initiating the slice cultures to avoid confounding the response to slicing with any drug response. PCLSs were evaluated after 24, 48, and 72 h exposures to NSC 710305. Reversibility of toxicity due to the 72-h treatment was evaluated after a 24-h recovery period. NSC 710305 caused a concentration-dependent cytokine response, and only the toxicity caused by a 72-h exposure to 25 µM reversed during the 24-h recovery period. Immuno/histological examination and quantitation of tissue protein levels indicated that tissue destruction, ED-1 (activated macrophage) staining, and protein levels were associated with the levels of proinflammatory cytokines in the tissue. In conclusion, the concentration- and time-dependent inflammatory response of PCLS to NSC 710305 preceded relevant tissue damage by a few days. The no-observable adverse effect level (NOAEL) for 24, 48, and 72 h exposures was established as 10 µM NSC 710305.