Evaluation of novel acute urinary rat kidney toxicity biomarker for subacute toxicity studies in preclinical trials.

Novel urinary protein biomarkers for the detection of acute renal damage, recently accepted by the U.S. Food and Drug Administration, European Medicines Agency, and Pharmaceuticals and Medical Devices Agency (Japan), now have to be validated in practice. Limited data regarding the performance of these acute markers after subacute or subchronic treatment are publicly available. To increase the area of applicability of these markers, it is important to evaluate the ability to detect them after 28 days of treatment or even longer. Wistar rats were treated with three doses of cisplatin, vancomycin, or puromycin to induce renal damage. Twelve candidate proteins were measured by Luminex xMAP-based WideScreen assays, MesoScale Discovery-based MULTI-SPOT technology, or RENA-strip dipstick assay after 28 days. Treatment with all three model compounds resulted in a dose-dependent increase in urinary biomarkers, specific for the observed areas within the nephron, determined histopathologically. The most promising biomarkers in this study were NGAL, Kim-1, osteopontin, clusterin, RPA-1, and GSTYb1, detected by multiplexing technologies. The RENA-strip dipstick assay delivered good diagnostic results for vancomycin-treated but not for cisplatin- or puromycin-treated rats. Taken together, the data show that these new biomarkers are robust and measurable for longer term studies to predict different types of kidney toxicities.

L-citrulline: A preclinical safety biomarker for the small intestine in rats and dogs in repeat dose toxicity studies.

INTRODUCTION: Gastrointestinal (GI) toxicity is still an issue within drug development, especially for novel oncology drugs. The identification of GI mucosal damage at an early stage with high sensitivity and specificity across preclinical species and humans remains difficult. To date, in preclinical studies, no qualified mechanistic, diagnostic or prognostic biomarkers exist for GI mucosal toxicity. L-citrulline is one of the most promising biomarker candidates used in clinical settings to quantify enterocyte integrity in various small intestinal diseases. L-citrulline is an intermediate metabolic amino acid produced mainly by functional enterocytes of the small intestine, whereby enterocyte loss will cause a drop in circulating L-citrulline. METHODS: In several repeat-dose toxicity studies, plasma L-citrulline has been evaluated as a potential safety biomarker for intestinal toxicity in beagle dogs and Wistar (Han) rats treated with different oncological drug candidates in drug development. Clinical observations and body weight determinations were performed during the pretreatment, treatment and treatment-free recovery period as well as toxicokinetic, gross and histopathology examinations. The quantitative determination of plasma L-citrulline levels during the pretreatment (only dogs), treatment and treatment-free recovery period were performed using an HPLC MS/MS assay. In cynomolgus monkeys, the first investigations on baseline L-citrulline levels were performed. RESULTS: In dogs, a dose- and exposure-dependent decrease of up to 50% in plasma L-citrulline was seen without histopathological alterations. However, a decrease of more than 50% in comparison to the individual animal pretreatment value of L-citrulline correlated very well with histopathological findings (intestinal crypt necrosis, villus atrophy, enterocyte loss) and clinical signs (bloody faeces and diarrhoea). During a treatment-free recovery period, a trend of increasing levels was observed in dogs. In rats, absolute L-citrulline plasma levels of treated animals decreased compared to the values of the concurrent control group. This decrease also correlated with the histopathological findings in the small intestine (single cell necrosis and mucosa atrophy). Because of a large physiological variation in L-citrulline plasma levels in dogs and rats, a clear cut-off value for absolute L-citrulline levels predictive of intestinal mucosal toxicity was difficult to establish. However, a > 50% decrease in L-citrulline plasma levels during the treatment period strongly correlated with histopathological findings. DISCUSSION: Based on the performed analysis, a longitudinal investigation of L-citrulline plasma levels for individual animals in the control and treatment groups is essential and pretreatment values of L-citrulline levels in rodents would be highly informative. Overall, further cross-species comparison (Cynomolgus monkey, mouse) and implementation in clinical trials as exploratory biomarker is essential to foster the hypothesis and to understand completely the clinical relevance of L-citrulline as a small intestine biomarker.

l-citrulline: A preclinical safety biomarker for the small intestine in rats and dogs in repeat dose toxicity studies.

INTRODUCTION: Gastrointestinal (GI) toxicity is still an issue within drug development, especially for novel oncology drugs. The identification of GI mucosal damage at an early stage with high sensitivity and specificity across preclinical species and humans remains difficult. To date, in preclinical studies, no qualified mechanistic, diagnostic or prognostic biomarkers exist for GI mucosal toxicity. l-citrulline is one of the most promising biomarker candidates used in clinical settings to quantify enterocyte integrity in various small intestinal diseases. l-citrulline is an intermediate metabolic amino acid produced mainly by functional enterocytes of the small intestine, whereby enterocyte loss will cause a drop in circulating l-citrulline. METHODS: In several repeat-dose toxicity studies, plasma l-citrulline has been evaluated as a potential safety biomarker for intestinal toxicity in beagle dogs and Wistar (Han) rats treated with different oncological drug candidates in drug development. Clinical observations and body weight determinations were performed during the pretreatment, treatment and treatment-free recovery period as well as toxicokinetic, gross and histopathology examinations. The quantitative determination of plasma l-citrulline levels during the pretreatment (only dogs), treatment and treatment-free recovery period were performed using an HPLC MS/MS assay. In cynomolgus monkeys, the first investigations on baseline l-citrulline levels were performed. RESULTS: In dogs, a dose- and exposure-dependent decrease of up to 50% in plasma l-citrulline was seen without histopathological alterations. However, a decrease of more than 50% in comparison to the individual animal pretreatment value of l-citrulline correlated very well with histopathological findings (intestinal crypt necrosis, villus atrophy, enterocyte loss) and clinical signs (bloody faeces and diarrhoea). During a treatment-free recovery period, a trend of increasing levels was observed in dogs. In rats, absolute l-citrulline plasma levels of treated animals decreased compared to the values of the concurrent control group. This decrease also correlated with the histopathological findings in the small intestine (single cell necrosis and mucosa atrophy). Because of a large physiological variation in l-citrulline plasma levels in dogs and rats, a clear cut-off value for absolute l-citrulline levels predictive of intestinal mucosal toxicity was difficult to establish. However, a > 50% decrease in l-citrulline plasma levels during the treatment period strongly correlated with histopathological findings. DISCUSSION: Based on the performed analysis, a longitudinal investigation of l-citrulline plasma levels for individual animals in the control and treatment groups is essential and pretreatment values of l-citrulline levels in rodents would be highly informative. Overall, further cross-species comparison (Cynomolgus monkey, mouse) and implementation in clinical trials as exploratory biomarker is essential to foster the hypothesis and to understand completely the clinical relevance of l-citrulline as a small intestine biomarker.

Limitations of the colloidal silica method in mapping the endothelial plasma membrane proteome of the mouse heart.

The endothelial cell (EC) membrane is an important interface, which plays a crucial role in signal transduction. Our aim was to selectively purify luminal EC membrane proteins from the coronary vasculature of the isolated perfused mouse heart and analyze its composition with mass spectrometry (MS). To specifically label coronary ECs in the intact heart, the colloidal silica method was applied, which is based on the binding of positively charged colloidal silica to the surface of EC membranes. Transmission electron microscopy revealed the specific labeling of ECs of macro and microvessels. Two different methods of tissue homogenization (Teflon pestle and ultra blade) together with density centrifugation were used for membrane protein enrichment. Enrichment and purity was controlled by Western blot analysis using the EC-specific protein caveolin 1 and various intracellular marker proteins. The ultra blade method resulted in a tenfold enrichment of caveolin 1, while there was negligible contamination as judged by Western blot. However, protein yield was low and required pooling of ten hearts for MS. When enriched endothelial membrane proteins were digested with trypsin and analyzed by LC-MS, a total of 56 proteins could be identified, of which only 12 were membrane proteins. We conclude that coronary endothelial membranes can be conveniently labeled with colloidal silica. However, due to the ionic nature of interaction of colloidal silica with the EC membrane the shear rate required for cardiac homogenization resulted in a substantial loss of specificity.