Rat Urinary Osteopontin and Neutrophil Gelatinase-Associated Lipocalin Improve Certainty of Detecting Drug-Induced Kidney Injury.

Traditional kidney biomarkers are insensitive indicators of acute kidney injury, with meaningful changes occurring late in the course of injury. The aim of this work was to demonstrate the diagnostic potential of urinary osteopontin (OPN) and neutrophil gelatinase-associated lipocalin (NGAL) for drug-induced kidney injury (DIKI) in rats using data from a recent regulatory qualification submission of translational DIKI biomarkers and to compare performance of NGAL and OPN to five previously qualified DIKI urinary biomarkers. Data were compiled from 15 studies of 11 different pharmaceuticals contributed by Critical Path Institute's Predictive Safety Testing Consortium (PSTC) Nephrotoxicity Working Group (NWG). Rats were given doses known to cause DIKI or other target organ toxicity, and urinary levels of the candidate biomarkers were assessed relative to kidney histopathology and serum creatinine (sCr) and blood urea nitrogen (BUN).OPN and NGAL outperformed sCr and BUN in identifying DIKI manifested as renal tubular epithelial degeneration or necrosis. In addition, urinary OPN and NGAL, when used with sCr and BUN, increased the ability to detect renal tubular epithelial degeneration or necrosis. NGAL and OPN had comparable or improved performance relative to Kim-1, clusterin, albumin, total protein, and beta-2 microglobulin. Given these data, both urinary OPN and NGAL are appropriate for use with current methods for assessing nephrotoxicity to identify and monitor DIKI in regulatory toxicology studies in rats. These data also support exploratory use of urinary OPN and NGAL in safety monitoring strategies of early clinical trials to aid in the assurance of patient safety.

CNS amyloid-ß, soluble APP-α and -ß kinetics during BACE inhibition.

BACE, a ß-secretase, is an attractive potential disease-modifying therapeutic strategy for Alzheimer's disease (AD) as it results directly in the decrease of amyloid precursor protein (APP) processing through the ß-secretase pathway and a lowering of CNS amyloid-ß (Aß) levels. The interaction of the ß-secretase and α-secretase pathway-mediated processing of APP in the rhesus monkey (nonhuman primate; NHP) CNS is not understood. We hypothesized that CNS inhibition of BACE would result in decreased newly generated Aß and soluble APPß (sAPPß), with increased newly generated sAPPα. A stable isotope labeling kinetics experiment in NHPs was performed with a (13)C6-leucine infusion protocol to evaluate effects of BACE inhibition on CNS APP processing by measuring the kinetics of sAPPα, sAPPß, and Aß in CSF. Each NHP received a low, medium, or high dose of MBI-5 (BACE inhibitor) or vehicle in a four-way crossover design. CSF sAPPα, sAPPß, and Aß were measured by ELISA and newly incorporated label following immunoprecipitation and liquid chromatography-mass spectrometry. Concentrations, kinetics, and amount of newly generated APP fragments were calculated. sAPPß and sAPPα kinetics were similar, but both significantly slower than Aß. BACE inhibition resulted in decreased labeled sAPPß and Aß in CSF, without observable changes in labeled CSF sAPPα. ELISA concentrations of sAPPß and Aß both decreased and sAPPα increased. sAPPα increased by ELISA, with no difference by labeled sAPPα kinetics indicating increases in product may be due to APP shunting from the ß-secretase to the α-secretase pathway. These results provide a quantitative understanding of pharmacodynamic effects of BACE inhibition on NHP CNS, which can inform about target development.

The use of SSMD-based false discovery and false nondiscovery rates in genome-scale RNAi screens.

In genome-scale RNA interference (RNAi) screens, it is critical to control false positives and false negatives statistically. Traditional statistical methods for controlling false discovery and false nondiscovery rates are inappropriate for hit selection in RNAi screens because the major goal in RNAi screens is to control both the proportion of short interfering RNAs (siRNAs) with a small effect among selected hits and the proportion of siRNAs with a large effect among declared nonhits. An effective method based on strictly standardized mean difference (SSMD) has been proposed for statistically controlling false discovery rate (FDR) and false nondiscovery rate (FNDR) appropriate for RNAi screens. In this article, the authors explore the utility of the SSMD-based method for hit selection in RNAi screens. As demonstrated in 2 genome-scale RNAi screens, the SSMD-based method addresses the unmet need of controlling for the proportion of siRNAs with a small effect among selected hits, as well as controlling for the proportion of siRNAs with a large effect among declared nonhits. Furthermore, the SSMD-based method results in reasonably low FDR and FNDR for selecting inhibition or activation hits. This method works effectively and should have a broad utility for hit selection in RNAi screens with replicates.

Hit selection with false discovery rate control in genome-scale RNAi screens.

RNA interference (RNAi) is a modality in which small double-stranded RNA molecules (siRNAs) designed to lead to the degradation of specific mRNAs are introduced into cells or organisms. siRNA libraries have been developed in which siRNAs targeting virtually every gene in the human genome are designed, synthesized and are presented for introduction into cells by transfection in a microtiter plate array. These siRNAs can then be transfected into cells using high-throughput screening (HTS) methodologies. The goal of RNAi HTS is to identify a set of siRNAs that inhibit or activate defined cellular phenotypes. The commonly used analysis methods including median +/- kMAD have issues about error rates in multiple hypothesis testing and plate-wise versus experiment-wise analysis. We propose a methodology based on a Bayesian framework to address these issues. Our approach allows for sharing of information across plates in a plate-wise analysis, which obviates the need for choosing either a plate-wise or experimental-wise analysis. The proposed approach incorporates information from reliable controls to achieve a higher power and a balance between the contribution from the samples and control wells. Our approach provides false discovery rate (FDR) control to address multiple testing issues and it is robust to outliers.

Inhibition of retinal and choroidal neovascularization by a novel KDR kinase inhibitor.

PURPOSE: Inhibition of vascular endothelial growth factor (VEGF) signaling has shown great promise for the treatment of ocular neovascular disease. Current anti-VEGF therapies in late-stage development, while efficacious, require dosing by frequent intravitreal injections that are inconvenient to patients. VEGF signaling inhibitors that demonstrate more convenient dosing regimens could lead to the improved treatment of neovascular diseases such as wet age related macular degeneration (AMD) and proliferative diabetic retinopathy (PDR). Here we describe the assessment of a KDR (VEGFR2) kinase inhibitor in two well-established models of ocular neovascularization following oral administration. METHODS: A novel KDR kinase inhibitor was dosed by oral gavage for 12 days at 0, 10, 30, or 100 mg/kg in an adult male Brown Norway rat laser induced choroidal neovascularization (CNV) model. The areas of CNV lesions were quantitated by fluorescence image analysis of FITC-dextran perfused animals. The kinase inhibitor was also assessed in a rat oxygen induced retinopathy (OIR) model in which neonatal rats were placed in an oxygen chamber that delivered alternating 24 h cycles of 50% and 10% oxygen for 14 days. After 14 days of oxygen treatment, the animals were returned to room air and dosed orally for 7 days with 0, 10, or 30 mg/kg kinase inhibitor. The extent of retinal neovascularization was assessed by counting pre-retinal neovascular nuclei on histological sections. RESULTS: At doses of 100 mg/kg, the KDR kinase inhibitor resulted in a 98% reduction in lesion size in the rat CNV model. 30 mg/kg doses of the inhibitor showed a 70% and 80% reduction in lesion size in the laser CNV and OIR models, respectively. CONCLUSIONS: Oral dosing of the described KDR kinase inhibitor effectively inhibits neovascularization in two well-established animal models of ocular neovascularization. These data suggest that compounds of this class may prove to be useful for the treatment of a variety of ocular neovascular diseases using a convenient oral dosing regimen.

Regulation of gene expression by lithium and depletion of inositol in slices of adult rat cortex.

Lithium inhibits inositol monophosphatase at therapeutically effective concentrations, and it has been hypothesized that depletion of brain inositol levels is an important chemical alteration for lithium's therapeutic efficacy in bipolar disorder. We have employed adult rat cortical slices as a model to investigate the gene regulatory consequences of inositol depletion effected by lithium using cytidine diphosphoryl-diacylglycerol as a functionally relevant biochemical marker to define treatment conditions. Genes coding for the neuropeptide hormone pituitary adenylate cyclase activating polypeptide (PACAP) and the enzyme that processes PACAP's precursor to the mature form, peptidylglycine alpha-amidating monooxygenase, were upregulated by inositol depletion. Previous work has shown that PACAP can increase tyrosine hydroxylase (TH) activity and dopamine release, and we found that the gene for GTP cyclohydrolase, which effectively regulates TH through synthesis of tetrahydrobiopterin, was also upregulated by inositol depletion. We propose that modulation of brain PACAP signaling might represent a new opportunity in the treatment of bipolar disorder.

Treatment with indinavir, efavirenz, and adefovir after failure of nelfinavir therapy.

A prospective, open-label study was conducted to assess the response to indinavir, efavirenz, and adefovir in human immunodeficiency virus (HIV)-infected patients experiencing viral rebound while receiving therapy with nelfinavir-containing regimens, to determine whether the protease genotype influenced the outcome of the salvage regimen. Genotyping from 29 nelfinavir failures revealed D30N in 17 (59%) and L90M in 11 (38%) cases. Suppression to <400 viral RNA copies/mL was achieved at week 48 in 56% of patients with the D30N virus versus 18% of patients with the L90M virus.