Translational safety biomarkers of colonic barrier integrity in the rat.

The intestinal barrier controls intestinal permeability, and its disruption has been associated with multiple diseases. Therefore, preclinical safety biomarkers monitoring barrier integrity are essential during the development of drugs targeting the intestines, particularly if starting treatment early after onset of disease. Classical toxicology endpoints are not sensitive enough and therefore our objective was to identify non-invasive markers enabling early in vivo detection of colonic barrier perturbation. Male Sprague-Dawley rats were dosed intracolonically via the rectum, using sodium caprate or ibuprofen as tool compounds to alter barrier integrity. Several potentially translational biomarkers and probe molecules related to permeability, inflammation or tissue damage were evaluated, using various analytical platforms, including immunoassays, targeted metabolomics and highly sensitive ultra-performance liquid chromatography-tandem mass spectrometry. Several markers were identified that allow early in vivo detection of colonic barrier integrity changes, before histopathological evidence of tissue damage. The most promising permeability markers identified were plasma fluorescein isothiocyanate-dextran 4000 and a lactulose/mannitol/sucralose mixture in urine. These markers showed maximum increases over 100-fold or approximately 10-50-fold, respectively. Intracolonic administration of the above probe molecules outperformed oral administration and inflammatory or other biomarkers, such as α2 -macroglobulin, calprotectin, cytokines, prostaglandins and a panel of metabolic molecules to identify early and subtle changes in barrier integrity. However, optimal timing of probe administration and sample collection is important for all markers evaluated. Inclusion of these probe molecules in preclinical toxicity studies might aid in risk assessment and the design of a clinical biomarker plan, as several of these markers have translational potential.

Analytical performance of a commercial multiplex Luminex-based cytokine panel in the rat.

INTRODUCTION: Multiplex immunoassays are an important tool in biomarker research during preclinical drug development. However, information regarding analytical performance of commercial multiplex assays for animal species is often limited. To be able to correctly interpret study results, a fit-for-purpose validation approach is recommended. The objective of our study was to provide a realistic example of what level of validation can be expected from this type of assay, using a rat cytokine panel. METHODS: The analytical performance of a commercial Luminex-based multiplex assay comprising IFN-γ, IL-6, IL-10, IL-12p70, IP-10 and TNF-α was evaluated in Sprague-Dawley rat plasma and serum. Calibration curve, working range, precision, accuracy, selectivity, parallelism, dilutional linearity, prozone effect and sample stability were assessed. RESULTS: Analytical performance in plasma and serum was comparable. Precision and accuracy results for all analytes were acceptable with coefficient of variation (CV) and relative error (RE) often below 15%, except for serum IL-6. Selectivity results varied per analyte with several cytokines showing CV>30% and no single minimum required dilution (MRD) could be identified. In addition, some striking differences between recombinant and endogenous protein results were observed. A pronounced prozone effect was detected for IP-10. Analytes in samples stored at -70°C were stable (RE<30%) from 1 up to 6months depending on the analyte. DISCUSSION: The results illustrate the challenges encountered during validation of commercial animal Luminex-based multiplex assays, revealing analytical limitations such as matrix and prozone effects. The Milliplex rat cytokine panel under investigation was deemed suitable for relative quantification of exploratory type biomarkers.

Chronic drug-induced effects on contractile motion properties and cardiac biomarkers in human induced pluripotent stem cell-derived cardiomyocytes.

BACKGROUND AND PURPOSE: In the pharmaceutical industry risk assessments of chronic cardiac safety liabilities are mostly performed during late stages of preclinical drug development using in vivo animal models. Here, we explored the potential of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) to detect chronic cardiac risks such as drug-induced cardiomyocyte toxicity. EXPERIMENTAL APPROACH: Video microscopy-based motion field imaging was applied to evaluate the chronic effect (over 72 h) of cardiotoxic drugs on the contractile motion of hiPS-CMs. In parallel, the release of cardiac troponin I (cTnI), heart fatty acid binding protein (FABP3) and N-terminal pro-brain natriuretic peptide (NT-proBNP) was analysed from cell medium, and transcriptional profiling of hiPS-CMs was done at the end of the experiment. KEY RESULTS: Different cardiotoxic drugs altered the contractile motion properties of hiPS-CMs together with increasing the release of cardiac biomarkers. FABP3 and cTnI were shown to be potential surrogates to predict cardiotoxicity in hiPS-CMs, whereas NT-proBNP seemed to be a less valuable biomarker. Furthermore, drug-induced cardiotoxicity produced by chronic exposure of hiPS-CMs to arsenic trioxide, doxorubicin or panobinostat was associated with different profiles of changes in contractile parameters, biomarker release and transcriptional expression. CONCLUSION AND IMPLICATIONS: We have shown that a parallel assessment of motion field imaging-derived contractile properties, release of biomarkers and transcriptional changes can detect diverse mechanisms of chronic drug-induced cardiac liabilities in hiPS-CMs. Hence, hiPS-CMs could potentially improve and accelerate cardiovascular de-risking of compounds at earlier stages of drug discovery. LINKED ARTICLES: This article is part of a themed section on New Insights into Cardiotoxicity Caused by Chemotherapeutic Agents. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.21/issuetoc.

Cross-laboratory analytical validation of the cardiac biomarker NT-proANP in rat.

INTRODUCTION: Natriuretic peptides, including N-terminal-proatrial natriuretic peptide (NT-proANP) are cardiac hormones that are produced in response to myocardial stretch and have been used in rats and humans as blood based functional cardiac biomarkers. There are limited validation data of these assays in rats and therefore the Predictive Safety Testing Consortium, Cardiac Hypertrophy Working Group (PSTC-CHWG) performed a cross-laboratory (5 laboratories) analytical evaluation of a commercially available NT-proANP ELISA for use with rat samples. METHODS: Serum samples were collected from normal Sprague Dawley (SD) rats and were spiked with kit calibrator material or rat heart tissue extracts to provide specimens for the validation. In addition, the cardiotoxicant, isoproterenol, was used to induce elevated endogenous NT-proANP levels in a subgroup of rats for additional validation specimens. The Biomedica™ (BI-20892, Vienna, Austria) proANP (1-98) enzyme-linked immunoabsorbent assay (ELISA) kit was used to measure NT-proANP. Intra-assay and inter-assay precisions, accuracy, sample linearity, recovery, limit of detection, upper and lower limits of quantitation (ULOQ and LLOQ, respectively), sample-freeze/thaw stability and stored sample stability were assessed and compared to pre-determined acceptance criteria. RESULTS: The majority of the experimental assessments met the established validation criteria, however there were individual results that did not meet these standards. Overall, acceptable intra- and inter-assay precisions and accuracies as well as inter-laboratory precision and accuracy were demonstrated. Linearity and recovery values fell within the pre-determined acceptance criteria, samples remained stable for up to three freeze-thaw cycles and frozen samples were stable at ~-70 °C for 12 months. The limit of detection (LOD) and LLOQ and ULOQ were similar to those specified by the manufacturer. DISCUSSION: Overall, the assay was demonstrated to be technically adequate for the detection of NT-proANP serum levels in SD rats.

Tissue Kim-1 and urinary clusterin as early indicators of cisplatin-induced acute kidney injury in rats.

The kidney is one of the main targets of drug toxicity, and early detection of renal damage is critical in preclinical drug development. A model of cisplatin-induced nephrotoxicity in male Sprague Dawley rats treated for 1, 3, 5, 7, or 14 days at 1 mg/kg/day was used to monitor the spatial and temporal expression of various indicators of kidney toxicity during the progression of acute kidney injury (AKI). As early as 1 day after cisplatin treatment, positive kidney injury molecule-1 (Kim-1) immunostaining, observed in the outer medulla of the kidney, and changes in urinary clusterin indicated the onset of proximal tubular injury in the absence of functional effects. After 3 days of treatment, Kim-1 protein levels in urine increased more than 20-fold concomitant with a positive clusterin immunostaining and an increase in urinary osteopontin. Tubular basophilia was also noted, while serum creatinine and blood urea nitrogen levels were elevated only after 5 days, together with tubular degeneration. In conclusion, tissue Kim-1 and urinary clusterin were the most sensitive biomarkers for detection of cisplatin-induced kidney damage. Thereafter, urinary Kim-1 and osteopontin, as well as clusterin immunostaining accurately correlated with the histopathological findings. When AKI is suspected in preclinical rat studies, Kim-1, clusterin, and osteopontin should be part of urinalysis and/or IHC can be performed.

Modeling anti-KLH ELISA data using two-stage and mixed effects models in support of immunotoxicological studies.

During preclinical drug development, the immune system is specifically evaluated after prolonged treatment with drug candidates, because the immune system may be an important target system. The response of antibodies against a T-cell-dependent antigen is recommenced by the FDA and EMEA for the evaluation of immunosuppression/enhancement. For that reason, we developed a semiquantitative enzyme-linked immunosorbent assay to measure antibodies against keyhole limpet hemocyanin. To our knowledge, the analysis of this kind of data is at this moment not yet fully explored. In this article, we describe two approaches for modeling immunotoxic data using nonlinear models. The first is a two-stage model in which we fit an individual nonlinear model for each animal in the first stage, and the second stage consists of testing possible treatment effects using the individual maximum likelihood estimates obtained in the first stage. In the second approach, the inference about treatment effects is based on a nonlinear mixed model, which accounts for heterogeneity between animals. In both approaches, we use a three-parameter logistic model for the mean structure.

MODELING ANTI-KLH ELISA DATA USING TWO-STAGE AND MIXED EFFECTS MODELS IN SUPPORT OF IMMUNOTOXICOLOGICAL STUDIES.

During preclinical drug development, the immune system is specifically evaluated after prolonged treatment with drug candidates, because the immune system may be an important target system. The response of antibodies against a T-cell-dependent antigen is recommenced by the FDA and EMEA for the evaluation of immunosuppression/enhancement. For that reason, we developed a semiquantitative enzyme-linked immunosorbent assay to measure antibodies against keyhole limpet hemocyanin. To our knowledge, the analysis of this kind of data is at this moment not yet fully explored. In this article, we describe two approaches for modeling immunotoxic data using nonlinear models. The first is a two-stage model in which we fit an individual nonlinear model for each animal in the first stage, and the second stage consists of testing possible treatment effects using the individual maximum likelihood estimates obtained in the first stage. In the second approach, the inference about treatment effects is based on a nonlinear mixed model, which accounts for heterogeneity between animals. In both approaches, we use a three-parameter logistic model for the mean structure.