Approved and marketed drugs are frequently studied in nonclinical models to evaluate the potential application to additional disease indications or to gain insight about molecular mechanisms of action. A survey of the literature reveals that nonclinical experimental designs (in vitro or in vivo) often include evaluation of drug concentrations or doses that are much higher than what can be achieved in patients (i.e., above the maximally tolerated dose or much higher than the clinically relevant exposures). The results obtained with these high concentrations may be particularly helpful in elucidating off-target effects and toxicities, but it is critical to have a dose-response curve that includes the minimally effective or clinically effective concentration for comparison. We have reviewed the clinical literature and drug product labels for all small molecules and biological agents approved by the U.S. Food and Drug Administration (FDA) for use in oncology in order to identify and compile the available pharmacokinetic parameters. The data summarized here can serve as a guide for selection of in vitro concentrations and in vivo plasma exposures for evaluation of drug effects in nonclinical studies. Inclusion of drug concentrations or exposures that are relevant to those observed in clinical practice can improve translation of nonclinical mechanism of action findings into potentially relevant clinical effects.
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 Consortium's (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.
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 plethora of novel molecular-targeted agents (MTAs) has provided an opportunity to selectively target pathways involved in carcinogenesis and tumour progression. Combination strategies of MTAs are being used to inhibit multiple aberrant pathways in the hope of optimizing antitumour efficacy and to prevent development of resistance. While the selection of specific agents in a given combination has been based on biological considerations (including the role of the putative targets in cancer) and the interactions of the agents used in combination, there has been little exploration of the possible enhanced toxicity of combinations resulting from alterations in multiple signalling pathways in normal cell biology. Owing to the complex networks and crosstalk that govern normal and tumour cell proliferation, inhibiting multiple pathways with MTA combinations can result in unpredictable disturbances in normal physiology. This Review focuses on the main toxicities and the lack of tolerability of some common MTA combinations, particularly where evidence of enhanced toxicity compared to either agent alone is documented or there is development of unexpected toxicity. Toxicities caused by MTA combinations highlight the need to introduce new preclinical testing paradigms early in the drug development process for the assessment of chronic toxicities resulting from such combinations.
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.
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 structureactivity relationships, and prioritizing development of compounds with improved safety profiles early in drug discovery.
Chemotherapy-induced peripheral neuropathy (CIPN) is a major, dose-limiting adverse effect experienced by cancer patients. Advancements in mechanism-based risk mitigation and effective treatments for CIPN can be aided by suitable in vitro assays. To this end, we developed a multiparametric morphology-centered rat dorsal root ganglion (DRG) assay. Morphologic alterations in subcellular structures of neurons and non-neurons were analyzed with an automated microscopy system. Stains for NeuN (a neuron-specific nuclear protein) and Tuj-1 (β-III tubulin) were used to identify neuronal cell nuclei and neuronal cell bodies/neurites, respectively. Vimentin staining (a component of Schwann cell intermediate filaments) was used to label non-neuronal supporting cells. Nuclei that stained with DAPI, but lacked NeuN represented non-neuronal cells. Images were analyzed following 24 h of continuous exposure to CIPN-inducing agents and 72 h after drug removal to provide a dynamic measure of recovery from initial drug effects. Treatment with bortezomib, cisplatin, eribulin, paclitaxel or vincristine induced a dose-dependent loss of neurite/process areas, mimicking the 'dying back' degeneration of axons, a histopathological hallmark of clinical CIPN in vivo. The IC50 for neurite loss was within 3-fold of the maximal clinical exposure (Cmax) for all five CIPN-inducing drugs, but was >4- or ≥ 28-fold of the Cmax for 2 non-CIPN-inducing agents. Compound-specific effects, eg, neurite fragmentation by cisplatin or bortezomib and enlarged neuronal cell bodies by paclitaxel, were also observed. Collectively, these results support the use of a quantitative, morphologic evaluation and a DRG cell culture model to inform risk and examine mechanisms of CIPN.
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.
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