Acute silica toxicity: attenuation by amiodarone-induced pulmonary phospholipidosis.

Exposure to the toxic mineral dust silica has been shown to produce an acute inflammatory response in the lungs of both humans and laboratory animals. Coating silica with phospholipids reduces its toxicity when studied with in vitro systems. The drug amiodarone increases phospholipid within the cells, airways, and alveoli of the lungs. This increase in phospholipid is due to amiodarone\u27s ability to inhibit phospholipase activity within alveolar macrophages (AMs) and whole lung. The purpose of this study was to determine whether the amiodarone-induced increase in pulmonary phospholipid would protect the lungs from acute damage caused by the intratracheal instillation of silica. Treatment of male Fischer 344 rats with amiodarone for 14 days caused an increase in phospholipid content in bronchoalveolar lavage fluid and AMs compared to vehicle-treated controls. The rats were then instilled with silica or saline vehicle. At both 1 and 14 days after silica exposure, pulmonary phospholipidosis was associated with a marked reduction in acute silica-induced pulmonary damage as assessed by biochemical parameters in bronchoalveolar lavage fluid, however, the influx of neutrophils into the airspaces was not reduced. Four times more phospholipid was bound to the silica recovered from amiodarone-treated rats compared to controls. The results of these in vivo experiments indicate that pulmonary phospholipidosis attenuates the acute damage associated with the intratracheal instillation of silica in rats. By using an in vitro cell culture system, we demonstrated that, in contrast to control AMs, phospholipidotic AMs were significantly more resistant to the cytotoxicity of surfactant-coated silica.(ABSTRACT TRUNCATED AT 250 WORDS

Predicting Phospholipidosis Using Machine Learning

Phospholipidosis is an adverse effect caused by numerous cationic amphiphilic drugs and can affect many cell types. It is characterized by the excess accumulation of phospholipids and is most reliably identified by electron microscopy of cells revealing the presence of lamellar inclusion bodies. The development of phospholipidosis can cause a delay in the drug development process, and the importance of computational approaches to the problem has been well documented. Previous work on predictive methods for phospholipidosis showed that state of the art machine learning methods produced the best results. Here we extend this work by looking at a larger data set mined from the literature. We find that circular fingerprints lead to better models than either E-Dragon descriptors or a combination of the two. We also observe very similar performance in general between Random Forest and Support Vector Machine models.</p

Morphometric Characterization of Rat and Human Alveolar Macrophage Cell Models and their Response to Amiodarone using High Content Image Analysis

© The Author(s) 2017. This article is an open access publication. Open Access: This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.Purpose. Progress to the clinic may be delayed or prevented when vacuolated or “foamy” alveolar macrophages are observed during non-clinical inhalation toxicology assessment. The first step in developing methods to study this response in vitro is to characterize macrophage cell lines and their response to drug exposures.Methods. Human (U937) and rat (NR8383) cell lines and primary rat alveolar macrophages obtained by bronchoalveolar lavage were characterized using high content fluorescence imaging analysis quantification of cell viability, morphometry, and phospholipid and neutral lipid accumulation. Results. Cell health, morphology and lipid content were comparable (p<0.05) for both cell lines and the primary macrophages in terms of vacuole number, size and lipid content. Responses to amiodarone, a known inducer of phospholipidosis, required analysis of shifts in cell population profiles (the proportion of cells with elevated vacuolation or lipid content) rather than average population data which was insensitive to the changes observed.Conclusions. A high content image analysis assay was developed and used to provide detailed morphological characterization of rat and human alveolar-like macrophages and their response to a phospholipidosis-inducing agent. This provides a basis for development of assays to predict or understand macrophage vacuolation following inhaled drug exposure.Peer reviewedFinal Published versio

Preservation of quality of life in patients with human epidermal growth factor receptor 2–positive metastatic breast cancer treated with tucatinib or placebo when added to trastuzumab and capecitabine (HER2CLIMB trial)

AIMS: In HER2CLIMB, tucatinib significantly improved progression-free and overall survival in patients with human epidermal growth factor receptor 2–positive (HER2+) metastatic breast cancer. We evaluated the impact of tucatinib on health-related quality of life (HR-QoL) in HER2CLIMB. METHODS: Patients were randomised 2:1 to tucatinib or placebo combined with trastuzumab and capecitabine. Starting with protocol version 7, the EuroQol 5 Dimensions 5 Levels (EQ-5D-5L) questionnaire and EQ visual analogue scale (VAS) were administered at day 1 of cycle 1, every two cycles during cycles 3–9, every three cycles during cycle 12 and thereafter and at each patient's 30-day follow-up visit. RESULTS: Among 364 patients eligible for HR-QoL assessment, 331 (91%) completed ≥1 assessment. EQ-VAS scores were similar for both arms at baseline and maintained throughout treatment. EQ-5D-5L scores were similar between the treatment arms, stable throughout therapy and worsened after discontinuing treatment. Risk of meaningful deterioration (≥7 points) on EQ-VAS was reduced 19% in the tucatinib vs. placebo arm (hazard ratio [HR]: 0.81; 95% confidence interval [CI]: 0.55, 1.18); the median (95% CI) time to deterioration was not reached in the tucatinib arm and was 5.8 months (4.3, -) in the placebo arm. Among patients with brain metastases (n = 164), risk of meaningful deterioration on EQ-VAS was reduced 49% in the tucatinib arm (HR: 0.51; 95% CI: 0.28, 0.93); the median (95% CI) time to deterioration was not reached in the tucatinib arm and was 5.5 months (4.2, -) in the placebo arm. CONCLUSIONS: HR-QoL was preserved for patients with HER2+ metastatic breast cancer who were treated with tucatinib added to trastuzumab and capecitabine and maintained longer with tucatinib therapy than without it among those with brain metastases

Single-cell analysis: visualizing pharmaceutical and metabolite uptake in cells with label-free 3D mass spectrometry imaging

Detecting metabolites and parent compound within a cell type is now a priority for pharmaceutical development. In this context, three-dimensional secondary ion mass spectrometry (SIMS) imaging was used to investigate the cellular uptake of the antiarrhythmic agent amiodarone, a phospholipidosis-inducing pharmaceutical compound. The high lateral resolution and 3D imaging capabilities of SIMS combined with the multiplex capabilities of ToF mass spectrometric detection allows for the visualization of pharmaceutical compound and metabolites in single cells. The intact, unlabeled drug compound was successfully detected at therapeutic dosages in macrophages (cell line: NR8383). Chemical information from endogenous biomolecules was used to correlate drug distributions with morphological features. From this spatial analysis, amiodarone was detected throughout the cell with the majority of the compound found in the membrane and subsurface regions and absent in the nuclear regions. Similar results were obtained when the macrophages were doped with amiodarone metabolite, desethylamiodarone. The FWHM lateral resolution measured across an intracellular interface in a high lateral resolution ion images was approximately 550 nm. Overall, this approach provides the basis for studying cellular uptake of pharmaceutical compounds and their metabolites on the single cell level

Simulation-based cheminformatic analysis of organelle-targeted molecules: lysosomotropic monobasic amines

Cell-based molecular transport simulations are being developed to facilitate exploratory cheminformatic analysis of virtual libraries of small drug-like molecules. For this purpose, mathematical models of single cells are built from equations capturing the transport of small molecules across membranes. In turn, physicochemical properties of small molecules can be used as input to simulate intracellular drug distribution, through time. Here, with mathematical equations and biological parameters adjusted so as to mimic a leukocyte in the blood, simulations were performed to analyze steady state, relative accumulation of small molecules in lysosomes, mitochondria, and cytosol of this target cell, in the presence of a homogenous extracellular drug concentration. Similarly, with equations and parameters set to mimic an intestinal epithelial cell, simulations were also performed to analyze steady state, relative distribution and transcellular permeability in this non-target cell, in the presence of an apical-to-basolateral concentration gradient. With a test set of ninety-nine monobasic amines gathered from the scientific literature, simulation results helped analyze relationships between the chemical diversity of these molecules and their intracellular distributions

A metabolomics cell-based approach for anticipating and investigating drug-induced liver injury

In preclinical stages of drug development, anticipating potential adverse drug effects such as toxicity is an important issue for both saving resources and preventing public health risks. Current in vitro cytotoxicity tests are restricted by their predictive potential and their ability to provide mechanistic information. This study aimed to develop a metabolomic mass spectrometry-based approach for the detection and classification of drug-induced hepatotoxicity. To this end, the metabolite profiles of human derived hepatic cells (i.e., HepG2) exposed to different well-known hepatotoxic compounds acting through different mechanisms (i.e., oxidative stress, steatosis, phospholipidosis, and controls) were compared by multivariate data analysis, thus allowing us to decipher both common and mechanism-specific altered biochemical pathways. Briefly, oxidative stress damage markers were found in the three mechanisms, mainly showing altered levels of metabolites associated with glutathione and γ-glutamyl cycle. Phospholipidosis was characterized by a decreased lysophospholipids to phospholipids ratio, suggestive of phospholipid degradation inhibition. Whereas, steatosis led to impaired fatty acids β-oxidation and a subsequent increase in triacylglycerides synthesis. The characteristic metabolomic profiles were used to develop a predictive model aimed not only to discriminate between non-toxic and hepatotoxic drugs, but also to propose potential drug toxicity mechanism(s)

Effect of Pembrolizumab Plus Neoadjuvant Chemotherapy on Pathologic Complete Response in Women With Early-Stage Breast Cancer: An Analysis of the Ongoing Phase 2 Adaptively Randomized I-SPY2 Trial.

Importance: Approximately 25% of patients with early-stage breast cancer who receive (neo)adjuvant chemotherapy experience a recurrence within 5 years. Improvements in therapy are greatly needed. Objective: To determine if pembrolizumab plus neoadjuvant chemotherapy (NACT) in early-stage breast cancer is likely to be successful in a 300-patient, confirmatory randomized phase 3 neoadjuvant clinical trial. Design, Setting, and Participants: The I-SPY2 study is an ongoing open-label, multicenter, adaptively randomized phase 2 platform trial for high-risk, stage II/III breast cancer, evaluating multiple investigational arms in parallel. Standard NACT serves as the common control arm; investigational agent(s) are added to this backbone. Patients with ERBB2 (formerly HER2)-negative breast cancer were eligible for randomization to pembrolizumab between November 2015 and November 2016. Interventions: Participants were randomized to receive taxane- and anthracycline-based NACT with or without pembrolizumab, followed by definitive surgery. Main Outcomes and Measures: The primary end point was pathologic complete response (pCR). Secondary end points were residual cancer burden (RCB) and 3-year event-free and distant recurrence-free survival. Investigational arms graduated when demonstrating an 85% predictive probability of success in a hypothetical confirmatory phase 3 trial. Results: Of the 250 women included in the final analysis, 181 were randomized to the standard NACT control group (median [range] age, 47 [24.77] years). Sixty-nine women (median [range] age, 50 [27-71] years) were randomized to 4 cycles of pembrolizumab in combination with weekly paclitaxel followed by AC; 40 hormone receptor (HR)-positive and 29 triple-negative. Pembrolizumab graduated in all 3 biomarker signatures studied. Final estimated pCR rates, evaluated in March 2017, were 44% vs 17%, 30% vs 13%, and 60% vs 22% for pembrolizumab vs control in the ERBB2-negative, HR-positive/ERBB2-negative, and triple-negative cohorts, respectively. Pembrolizumab shifted the RCB distribution to a lower disease burden for each cohort evaluated. Adverse events included immune-related endocrinopathies, notably thyroid abnormalities (13.0%) and adrenal insufficiency (8.7%). Achieving a pCR appeared predictive of long-term outcome, where patients with pCR following pembrolizumab plus chemotherapy had high event-free survival rates (93% at 3 years with 2.8 years\u27 median follow-up). Conclusions and Relevance: When added to standard neoadjuvant chemotherapy, pembrolizumab more than doubled the estimated pCR rates for both HR-positive/ERBB2-negative and triple-negative breast cancer, indicating that checkpoint blockade in women with early-stage, high-risk, ERBB2-negative breast cancer is highly likely to succeed in a phase 3 trial. Pembrolizumab was the first of 10 agents to graduate in the HR-positive/ERBB2-negative signature. Trial Registration: ClinicalTrials.gov Identifier: NCT01042379