Application of genomics for identification of systemic toxicity triggers associated with VEGF-R inhibitors.

The key to the discovery of new pharmaceuticals is to develop molecules that interact with the intended target and minimize interaction with unintended molecular targets, therefore minimizing toxicity. This is aided by the use of various in vitro selectivity assays that are used to select agents most potent for the desired target. Typically, molecules from similar chemical series, with similar in vitro potencies, are expected to yield comparable in vivo pharmacological and toxicological profiles, predictive of target effects. However, in this study, we investigated the in vivo effects of two analogue compounds that similarly inhibit several receptor tyrosine kinases such as vascular endothelial growth factor receptor 1 (VEGFR/Flt1), vascular endothelial growth factor 2 (VEGFR2/kinase domain receptor/Flk-1), vascular endothelial growth factor receptor 3 (VEGFR3/Flt4), platelet-derived growth factor receptor (PDGFR), and Kit receptors, which bear similar chemical structures, have comparable potencies, but differ markedly in their rodent toxicity profiles. Global gene expression data were used to generate hypotheses regarding the existence of toxicity triggers that would reflect the perturbation of signaling in multiple organs such as the liver, adrenal glands, and the pancreas in response to compound treatment. We concluded that differences in pharmacokinetic properties of the two analogues, such as volume of distribution, half-life, and organ concentrations, resulted in marked differences in the chemical burden on target organs and may have contributed to the vast differences in toxicity profiles observed with the two otherwise similar molecules. We propose including select toxicokinetic parameters such as V(ss), T(1/2), and T(max) as additional criteria that could be used to rank order compounds from the same pharmacological series to possibly minimize organ toxicity. Assessment of toxicokinetics is not an atypical activity on toxicology studies, even in early screening studies; however, these data may not always be used in decision making for selecting or eliminating one compound over another. Finally, we illustrate that in vivo gene expression profiles can serve as a complementary assessor of this activity and simultaneously help provide an assessment of on or off-target biological activity.

Assessment of repeated microarray experiments using mixed tissue RNA reference samples.

Genome-scale gene expression technologies are increasingly being applied for biological research as a whole and toxicological screening in particular. In order to monitor data quality and process drift, we adopted the use of two rat-tissue mixtures (brain, liver, kidney, and testis) previously introduced as RNA reference samples. These samples were processed every time a microarray experiment was hybridized, thereby verifying the comparability of the resulting expression data for cross-study comparison. This study presents the analysis of 21 technical replicates of these two mixed-tissue samples using Affymetrix RAE230_2 GeneChip over a period of 12 months. The results show that detection sensitivity, measured by the number of present and absent sequences, is robust, and data correlation, indicated by scatter plots, varies little over time. Receiver operating characteristic (ROC) curves show the sensitivity and specificity of the current measurements are consistent with arrays previously classified as well performing. Overall, this paper shows that the inclusion of standard samples during microarray labeling and hybridization experiments is useful to benchmark the performance of microarray experiments over time and allows discovery of any process drift that, if it occurs, may confound the comparison of these datasets.

Gene expression analysis in a canine model of X-linked Alport syndrome.

Chronic kidney disease (CKD) often culminates in renal failure as a consequence of progressive interstitial fibrosis and is an important cause of illness and death in dogs. Identification of disease biomarkers and gene expression changes will yield valuable information regarding the specific biological pathways involved in disease progression. Toward these goals, gene expression changes in the renal cortex of dogs with X-linked Alport syndrome (XLAS) were examined using microarray technology. Extensive changes in inflammatory, metabolic, immune, and extracellular matrix biology were revealed in affected dogs. Statistical analysis showed 133 genes that were robustly induced or repressed in affected animals relative to age-matched littermates. Altered expression of numerous major histocompatibility complex (MHC) molecules suggests that the immune system plays a significant role in XLAS. Increased expression of COL4A1 and TIMP-1 at the end stage of disease supports the suggestion that expression increases in association with progression of fibrosis and confirms an observation of increased COL4A1 protein expression. Clusterin may function as one of the primary defenses of the renal cortex against progressive injury in dogs with XLAS, as demonstrated here by increased CLU gene expression. Cellular mechanisms that function during excess oxidative stress might also act to deter renal damage, as evidenced by alterations in gene expression of SOD1, ACO1, FDXR, and GPX1. This investigation provides a better understanding of interstitial fibrosis pathogenesis, and potential biomarkers for early detection, factors that are essential to discovering more effective treatments thereby reducing clinical illness and death due to CKD.

The fall in creatine levels and creatine kinase isozyme changes in the failing heart are reversible: complex post-transcriptional regulation of the components of the CK system.

Decreases in total creatine kinase (CK) activity and creatine [Cr] combine to limit the capacity of the failing heart to rapidly re-synthesize ATP (energy reserve). If the loss in energy reserve could be reversed, cardiac contractile reserve may be improved. Here we test whether these changes are reversible during recovery from heart failure. Left ventricular (LV) contractile function was measured in chronically instrumented conscious dogs with heart failure (CHF) induced by cardiac pacing for 3-4 weeks, and after recovery from heart failure (Recovery) (unpaced) for 5-6 weeks. LV contractile function and contractile reserve were depressed in CHF but returned to control in Recovery. CK capacity fell by 55% in CHF due to decreases in [Cr] (-39%) and CK activity (-25%), but was fully restored in Recovery. CK-B isozyme activity, protein (Western) and mRNA levels (real time PCR), respectively, were higher by 2-, 5.4- and 11-fold in CHF and higher by 3-, 2- and 2-fold in Recovery. CK-MM activity was decreased (-30%) in CHF but returned to normal levels during Recovery; CK-M protein was 30% lower in both CHF and Recovery even though there were no changes in mRNA levels. A similar pattern was found for mitochondrial CK (sMtCK). Deceases in CK activity and [Cr] in CHF are reversible. Decreases in CK-MM and sMtCK activities, but not the increases in CK-BB and CK-MB, also reversed. Neither the changes in protein nor mRNA levels for CK-B and CK-M correlated to their activities, suggesting that CK is under complex post-transcriptional regulation.

Down-regulation of alpha class glutathione S-transferase by interleukin-1beta in human intestinal epithelial cells (Caco-2) in culture.

The influence of pro-inflammatory cytokines on alpha class glutathione S-transferase A1 and A2 (GSTA1/A2) expression was examined in human colonic epithelial cells (Caco-2) in culture. Dose-dependent reductions in GSTA1/A2 mRNA, protein, and activity levels occurred in Caco-2 cells cultured in conditioned medium (CM) from lipopolysaccharide-stimulated murine monocyte-macrophage cells (RAW 264.7). Neutralizing anti-interleukin-1beta (IL-1beta) antibodies attenuated this repression of GSTA1/A2 expression by CM. Moreover, recombinant human IL-1beta reduced GSTalpha expression at the mRNA, protein, and activity levels in a dose-related fashion. Reduction of GSTA1/A2 mRNA levels by IL-1beta was attenuated by pretreatment with IL-1 receptor antagonist. GSTA1/A2 mRNA half-lives were similar in control and IL-1beta-treated cells, indicating that IL-1beta has no effect on mRNA stability. In reporter gene studies, IL-1beta caused a dose-related reduction of luciferase activity in Caco-2 cells transfected with the full-length GSTA1 promoter-luciferase construct. Using truncated constructs, IL-1beta responsiveness was mapped to a region 286 base pairs upstream to the coding region. Deletion of a hepatic nuclear factor 1 (HNF-1) site in this region abrogated the IL-1beta-mediated repression of GSTA1 promoter activity. These results demonstrate that IL-1beta down-regulates GSTA1/A2 expression in cultured human enterocytes by a transcriptional mechanism involving an HNF-1 site.

Adipsin, a biomarker of gastrointestinal toxicity mediated by a functional gamma-secretase inhibitor.

Functional gamma-secretase inhibitors (FGSIs) can block the cleavage of several transmembrane proteins including amyloid precursor protein (APP), and the cell fate regulator Notch-1. FGSIs, by inhibiting APP processing, block the generation of amyloid beta (Abeta) peptides and may slow the development of Alzheimer's disease. FGSIs used to inhibit APP processing may disrupt Notch processing, thus interfering with cell fate determination. Described herein is a FGSI-mediated gastrointestinal toxicity characterized by cell population changes in the ileum of rats, which are indicative of Notch signaling disruption. Microarray analysis of ileum from FGSI-treated rats revealed differential expression responses in a number of genes indicative of Notch signaling perturbation, including the serine protease adipsin. We were able to show that FGSI-treated rats had elevated levels of adipsin protein in gastrointestinal contents and feces, and by immunohistochemistry demonstrated that adipsin containing ileum crypt cells were increased in FGSI-treated rats. The mouse Adipsin proximal promoter contains a putative binding site for the Notch-induced transcriptional regulator Hes-1, which we demonstrate is able to bind Hes-1. Additional studies in 3T3-L1 preadipocytes demonstrate that this FGSI inhibits Hes-1 expression while up-regulating adipsin expression. Overexpression of Hes-1 was able to down-regulate adipsin expression and block pre-adipocyte differentiation. We propose that adipsin is a Hes-1-regulated gene that is de-repressed during FGSI-mediated disruption of Notch/Hes-1 signaling. Additionally, the aberrant expression of adipsin, and its presence in feces may serve as a noninvasive biomarker of gastrointestinal toxicity associated with perturbed Notch signaling.