Nick Goeminne

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.

Aromatase inhibition by the antifungal ketoconazole.

The aromatase inhibitory properties of the antifungal ketoconazole were compared with those of aminoglutethimide. In rat granulosa cells ketoconazole and aminoglutethimide showed IC50 values for aromatase inhibition of 2 X 10(-6) and 6 X 10(-7) M respectively. In the rat, in vivo, ketoconazole was 5 times less potent than aminoglutethimide. In young women, 400 mg of ketoconazole only marginally lowered plasma levels of estradiol-17 beta. It is concluded that ketoconazole is not a compound of choice for clinical use as an aromatase inhibitor.

Effects of combined and sequential treatment with tamoxifen and the aromatase inhibitor vorozole on 7,12-dimethylbenz(a) anthracene-induced mammary carcinoma in the rat.

Abstract The aromatase inhibitor vorozole dose-dependently inhibited the growth of dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in the rat. An oral dose of 5 mg/kg per day brought about growth inhibition and reduction of tumor multiplicity similar to that produced by ovariectomy. Tamoxifen (8 mg/kg per day) also reduced tumor growth, albeit to a lesser extent than did ovariectomy. Concomitant administration of varying doses of tamoxifen with the fully effective dose of vorozole (5 mg/kg per day) tended to be less effective than ovariectomy or vorozole alone. This is likely to be due to the estrogen-agonistic effects of tamoxifen. Combination of tamoxifen with the partially effective dose of vorozole (1 mg/kg per day) gave results comparable with those obtained for either of these compounds used in monotherapy. Combining tamoxifen with a marginally active low dose of vorozole (0.2 mg/kg per day) resulted in a minor additional growth inhibition as compared with that obtained with this dose of vorozole alone. Sequential treatment with tamoxifen (8 mg/kg per day) for 42 days and vorozole (5 mg/kg per day) for 42 days, and vice-versa, was performed with a drug-free interval of 14 days between treatments. Tumors regressing under vorozole therapy relapsed when subsequently treated with tamoxifen. In contrast, vorozole further reduced tumor volumes in rats previously treated with tamoxifen. Furthermore, monotherapy with tamoxifen as well as the two sequential tamoxifen-vorozole treatment schedules were in most cases less effective than vorozole monotherapy in inhibiting both tumor growth and tumor multiplicity. Although extrapolation of these findings in cycling animals to the clinical situation, involving postmenopausal women, is not straightforward, these results warrant further studies in preclinical models. Moreover, clinical trials comparing the most effective aromatase inhibitors with tamoxifen in previously untreated postmenopausal patients with breast cancer may also be warranted.

Endocrine and Antitumoral Effects of R76713 in Rats

Some effects of daily oral administration of a new non-steroidal aromatase inhibitor on the pituitary-gonadal and adrenal functions were investigated in female rats. At doses of 1 mg/kg twice daily or higher, R 76713 lowered plasma estradiol levels to the range measured after ovariectomy Plasma progesterone levels and uterine weights decreased whilst LH levels increased but to a lesser extent than after ovariectomy. The other hormonal data show that long-term administration of R 76 713 does not modify the gluco- and mineralocorticoid hormone levels even at the highest dose studied (20 mg/kg, 4 h after treatment). Furthermore, both ovariectomy and R 76 713 treatment (1 and 5 mg/kg twice a day) induced almost complete regression of 9,12-dimethyl-1,2-benzanthracene-induced mammary carcinoma in rats. The appearance of new tumors during the treatment period was completely inhibited by R 76 713 whilst multiplicity of the remaining tumors was dramatically reduced.

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

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.

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.

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.

Translational safety biomarkers of colonic barrier integrity in the rat: RAT COLONIC BARRIER INTEGRITY BIOMARKERS

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.