Induction of hemangiosarcoma in mice after chronic treatment with S1P-modulator siponimod and its lack of relevance to rat and human.

A high incidence of hemangiosarcoma (HSA) was observed in mice treated for 2 years with siponimod, a sphingosine-1-phosphate receptor 1 (S1P1) functional antagonist, while no such tumors were observed in rats under the same treatment conditions. In 3-month rat (90 mg/kg/day) and 9-month mouse (25 and 75 mg/kg/day) in vivo mechanistic studies, vascular endothelial cell (VEC) activation was observed in both species, but VEC proliferation and persistent increases in circulating placental growth factor 2 (PLGF2) were only seen in the mouse. In mice, these effects were sustained over the 9-month study duration, while in rats increased mitotic gene expression was present at day 3 only and PLGF2 was induced only during the first week of treatment. In the mouse, the persistent VEC activation, mitosis induction, and PLGF2 stimulation likely led to sustained neo-angiogenesis which over life-long treatment may result in HSA formation. In rats, despite sustained VEC activation, the transient mitotic and PLGF2 stimuli did not result in the formation of HSA. In vitro, the mouse and rat primary endothelial cell cultures mirrored their respective in vivo findings for cell proliferation and PLGF2 release. Human VECs, like rat cells, were unresponsive to siponimod treatment with no proliferative response and no release of PLGF2 at all tested concentrations. Hence, it is suggested that the human cells also reproduce a lack of in vivo response to siponimod. In conclusion, the molecular mechanisms leading to siponimod-induced HSA in mice are considered species specific and likely irrelevant to humans.

The Utility of Gene Expression Profiling from Tissue Samples to Support Drug Safety Assessments.

Originally conceptualized as an integrated approach combining conventional toxicology methods with genome-wide expression profiling, toxicogenomics has promised to provide unequivocal relationships between the molecular changes elicited by a compound or a target pathway and the lesions that appear subsequently in the tissues. However, the discipline has only partially delivered on this promise, and the number of publications and submissions related to toxicogenomics is stagnating. The purpose of this article is to outline key factors contributing to a successful implementation of toxicogenomics in the drug discovery and development process. Paradigms and methods of toxicogenomics are briefly reviewed, and the prominence of biostatistics and its limitations in the particular context of nonclinical toxicology studies are discussed. We present specific approaches for pathophysiological contextualization of gene expression data derived from tissues with lesions at variable incidence and severity: "unmixing" (deconvolution) of molecular expression profiles from complex tissues, the invaluable contribution of reference data, the role of establishing causation between expression signals and pathologic changes (phenotypic anchoring), and especially molecular localization. These approaches compensate for the limitations of biostatistical analysis, which in turn, derive from tissue heterogeneity. Finally, impactful applications of toxicogenomics along the drug discovery and development process are exemplified, from the evaluation of potential target toxicities to the selection of candidate compounds and elucidation of the molecular and cellular mechanisms leading to chronic toxicity.

Heart structure-specific transcriptomic atlas reveals conserved microRNA-mRNA interactions.

MicroRNAs are short non-coding RNAs that regulate gene expression at the post-transcriptional level and play key roles in heart development and cardiovascular diseases. Here, we have characterized the expression and distribution of microRNAs across eight cardiac structures (left and right ventricles, apex, papillary muscle, septum, left and right atrium and valves) in rat, Beagle dog and cynomolgus monkey using microRNA sequencing. Conserved microRNA signatures enriched in specific heart structures across these species were identified for cardiac valve (miR-let-7c, miR-125b, miR-127, miR-199a-3p, miR-204, miR-320, miR-99b, miR-328 and miR-744) and myocardium (miR-1, miR-133b, miR-133a, miR-208b, miR-30e, miR-499-5p, miR-30e*). The relative abundance of myocardium-enriched (miR-1) and valve-enriched (miR-125b-5p and miR-204) microRNAs was confirmed using in situ hybridization. MicroRNA-mRNA interactions potentially relevant for cardiac functions were explored using anti-correlation expression analysis and microRNA target prediction algorithms. Interactions between miR-1/Timp3, miR-125b/Rbm24, miR-204/Tgfbr2 and miR-208b/Csnk2a2 were identified and experimentally investigated in human pulmonary smooth muscle cells and luciferase reporter assays. In conclusion, we have generated a high-resolution heart structure-specific mRNA/microRNA expression atlas for three mammalian species that provides a novel resource for investigating novel microRNA regulatory circuits involved in cardiac molecular physiopathology.

Investigative safety science as a competitive advantage for Pharma.

INTRODUCTION: Following a US National Academy of Sciences report in 2007 entitled "Toxicity Testing of the 21st Century: a Vision and a Strategy," significant advances within translational drug safety sciences promise to revolutionize drug discovery and development. The purpose of this review is to outline why investigative safety science is a competitive advantage for the pharmaceutical industry. AREAS COVERED: The article discusses the essential goals for modern investigative toxicologists including: cross-species target biology; molecular pathways of toxicity; and development of predictive tools, models and biomarkers that allow discovery researchers and clinicians to anticipate safety problems and plan ways to address them, earlier than ever before. Furthermore, the article emphasizes the importance of investigating unanticipated clinical safety signals through a combination of mechanistic preclinical studies and/or molecular characterization of clinical samples from affected organs. EXPERT OPINION: The traditional boundaries between pharma industry teams focusing on safety/efficacy and preclinical/clinical development are rapidly disappearing in favor of translational safety science-centric organizations with a vision of bringing more effective medicines forward safely and quickly. Comparative biology and mechanistic toxicology approaches facilitate: i) identifying translational safety biomarkers; ii) identifying new drug targets/indications; and iii) mitigating off-target toxicities. These value-adding safety science contributions will change traditional toxicologists from side-effect identifiers to drug development enablers.

Urinary clusterin, cystatin C, beta2-microglobulin and total protein as markers to detect drug-induced kidney injury.

Earlier and more reliable detection of drug-induced kidney injury would improve clinical care and help to streamline drug-development. As the current standards to monitor renal function, such as blood urea nitrogen (BUN) or serum creatinine (SCr), are late indicators of kidney injury, we conducted ten nonclinical studies to rigorously assess the potential of four previously described nephrotoxicity markers to detect drug-induced kidney and liver injury. Whereas urinary clusterin outperformed BUN and SCr for detecting proximal tubular injury, urinary total protein, cystatin C and beta2-microglobulin showed a better diagnostic performance than BUN and SCr for detecting glomerular injury. Gene and protein expression analysis, in-situ hybridization and immunohistochemistry provide mechanistic evidence to support the use of these four markers for detecting kidney injury to guide regulatory decision making in drug development. The recognition of the qualification of these biomarkers by the EMEA and FDA will significantly enhance renal safety monitoring.

Kidney injury molecule-1 outperforms traditional biomarkers of kidney injury in preclinical biomarker qualification studies.

Kidney toxicity accounts both for the failure of many drug candidates as well as considerable patient morbidity. Whereas histopathology remains the gold standard for nephrotoxicity in animal systems, serum creatinine (SCr) and blood urea nitrogen (BUN) are the primary options for monitoring kidney dysfunction in humans. The transmembrane tubular protein kidney injury molecule-1 (Kim-1) was previously reported to be markedly induced in response to renal injury. Owing to the poor sensitivity and specificity of SCr and BUN, we used rat toxicology studies to compare the diagnostic performance of urinary Kim-1 to BUN, SCr and urinary N-acetyl-beta-D-glucosaminidase (NAG) as predictors of kidney tubular damage scored by histopathology. Kim-1 outperforms SCr, BUN and urinary NAG in multiple rat models of kidney injury. Urinary Kim-1 measurements may facilitate sensitive, specific and accurate prediction of human nephrotoxicity in preclinical drug screens. This should enable early identification and elimination of compounds that are potentially nephrotoxic.

A panel of urinary biomarkers to monitor reversibility of renal injury and a serum marker with improved potential to assess renal function.

The Predictive Safety Testing Consortium's first regulatory submission to qualify kidney safety biomarkers revealed two deficiencies. To address the need for biomarkers that monitor recovery from agent-induced renal damage, we scored changes in the levels of urinary biomarkers in rats during recovery from renal injury induced by exposure to carbapenem A or gentamicin. All biomarkers responded to histologic tubular toxicities to varied degrees and with different kinetics. After a recovery period, all biomarkers returned to levels approaching those observed in uninjured animals. We next addressed the need for a serum biomarker that reflects general kidney function regardless of the exact site of renal injury. Our assay for serum cystatin C is more sensitive and specific than serum creatinine (SCr) or blood urea nitrogen (BUN) in monitoring generalized renal function after exposure of rats to eight nephrotoxicants and two hepatotoxicants. This sensitive serum biomarker will enable testing of renal function in animal studies that do not involve urine collection.

Monitoring kidney safety in drug development: emerging technologies and their implications.

Drug-induced kidney injury is a serious and not uncommon adverse event which needs to be considered during drug development. The current standards used to monitor kidney function, such as blood urea nitrogen and serum creatinine, are late indicators of kidney injury and thus do not allow for timely intervention before loss of function. Improving the diagnosis and monitoring of kidney damage goes hand-in-hand with the identification of new biomarkers and the development of technologies that enable their sensitive and specific measurements. In order to move beyond restriction to internal company decisions, every entity that demonstrates the qualities of a biomarker must gain acceptance by health authorities if it is to be used for regulatory decision making in preclinical studies and clinical trials. This review focuses on the most promising achievements of new technologies applied to monitoring drug-induced nephrotoxicity (eg, gene expression, imaging, in vitro screening, protein assays) and on the use and implications of peripheral biomarkers such as the urinary protein biomarkers glutathione S-transferase-alpha, N-acetyl-beta-d-glucosaminidase, total protein, cystatin C, beta2-microglobulin, KIM-1, lipocalin-2 and serum cystatin C. Finally, the associated regulatory processes for use in clinics are also discussed.

VeloceGenomics: an accelerated in vivo drug discovery approach to rapidly predict the biologic, drug-like activity of compounds, proteins, or genes.

PURPOSE: The aim of this study is to test the predictive power of in vivo multiorgan RNA expression profiling in identifying the biologic activity of molecules. METHODS: Animals were treated with compound A or B. At the end of the treatment period, in vivo multiorgan microarray-based gene expression data were collected. Investigators masked to the identity of the compounds analyzed the transcriptome signatures to define the molecular pathways affected by treatment and to hypothesize the biologic activity and potential therapeutic indications of the blinded compounds. RESULTS: For compound A, G-protein-coupled receptors and factors associated with cell growth were affected-growth hormone/insulin-like growth factor-1, glucagon/insulin axes, and general somatomedin-like activity. Deblinding showed the compound to be a somatostatin analog, SOM230, confirming the accuracy of the predicted biologic activity. For compound B, components of the inflammatory cascade potentially mediated by lipopolysaccharide, tumor necrosis factor, or proinflammatory cytokines were affected. The gene expression signatures were most consistent with an interleukin-6 family activity. Deblinding revealed that compound B was leukemia inhibitory factor. CONCLUSIONS: VeloceGenomics is a strategy of coupling in vivo compound testing with genomic technologies. The process enables prediction of the mechanism of action and, coupled with other relevant data, prediction of the suitability of compounds for advancement in the drug development process.

Pimecrolimus identifies a common genomic anti-inflammatory profile, is clinically highly effective in psoriasis and is well tolerated.

The ascomycin macrolactam pimecrolimus is a novel inflammatory cytokine release inhibitor that so far has not been administered systemically to humans. In this phase I/II randomized double-blind, placebo-controlled, multiple rising dose proof of concept study psoriasis patients were treated with oral pimecrolimus or placebo. Gene profiling identified a common genomic profile with a downregulation of genes associated with inflammation but no changes in gene expression linked to drug-related side-effects. A steady state of pimecrolimus was reached after 5-10 d, Cmax, and area under the curve (0-24) was 54.5 ng per ml and 589.9 ng h per ml, respectively, at steady state at the highest dose. There was clear clinical efficacy in patients receiving 20 mg pimecrolimus twice daily and 30 mg twice daily with a reduction of Psoriasis Area and Severity Index by 60% and 75%, respectively. Histopatho logically and immunopathologically there was a reversion of the psoriatic phenotype towards normal. There were no notable clinical, laboratory, kidney function, or immunologic side-effects. We conclude that pimecrolimus taken orally is highly effective in a concentration-dependent manner in patients with psoriasis and on a short-term basis it is well tolerated and this is confirmed by its pharmacogenomic profile. The latter also indicates that pimecrolimus should be equally effective in other inflammatory skin diseases.