NMR and MS Methods for Metabolomics.

Metabolomics, also often referred as "metabolic profiling," is the systematic profiling of metabolites in biofluids or tissues of organisms and their temporal changes. In the last decade, metabolomics has become more and more popular in drug development, molecular medicine, and other biotechnology fields, since it profiles directly the phenotype and changes thereof in contrast to other "-omics" technologies. The increasing popularity of metabolomics has been possible only due to the enormous development in the technology and bioinformatics fields. In particular, the analytical technologies supporting metabolomics, i.e., NMR, UPLC-MS, and GC-MS, have evolved into sensitive and highly reproducible platforms allowing the determination of hundreds of metabolites in parallel. This chapter describes the best practices of metabolomics as seen today. All important steps of metabolic profiling in drug development and molecular medicine are described in great detail, starting from sample preparation to determining the measurement details of all analytical platforms, and finally to discussing the corresponding specific steps of data analysis.

Functional inhibition of acid sphingomyelinase by Fluphenazine triggers hypoxia-specific tumor cell death.

Owing to lagging or insufficient neo-angiogenesis, hypoxia is a feature of most solid tumors. Hypoxic tumor regions contribute to resistance against antiproliferative chemotherapeutics, radiotherapy and immunotherapy. Targeting cells in hypoxic tumor areas is therefore an important strategy for cancer treatment. Most approaches for targeting hypoxic cells focus on the inhibition of hypoxia adaption pathways but only a limited number of compounds with the potential to specifically target hypoxic tumor regions have been identified. By using tumor spheroids in hypoxic conditions as screening system, we identified a set of compounds, including the phenothiazine antipsychotic Fluphenazine, as hits with novel mode of action. Fluphenazine functionally inhibits acid sphingomyelinase and causes cellular sphingomyelin accumulation, which induces cancer cell death specifically in hypoxic tumor spheroids. Moreover, we found that functional inhibition of acid sphingomyelinase leads to overactivation of hypoxia stress-response pathways and that hypoxia-specific cell death is mediated by the stress-responsive transcription factor ATF4. Taken together, the here presented data suggest a novel, yet unexplored mechanism in which induction of sphingolipid stress leads to the overactivation of hypoxia stress-response pathways and thereby promotes their pro-apoptotic tumor-suppressor functions to specifically kill cells in hypoxic tumor areas.

Characterization of a Novel Hafnium-Based X-ray Contrast Agent.

OBJECTIVE: Characterization of BAY-576, a new x-ray contrast agent which is not based on iodine, but rather on the heavy metal hafnium. Compared with iodine, hafnium provides better x-ray absorption in the energy range of computed tomography (CT) and allows images of comparable quality to be acquired at a significantly reduced radiation dose. MATERIALS AND METHODS: A range of standard methods were used to explore the physicochemistry of BAY-576 as well as its tolerability in in vitro assays, its pharmacokinetics and toxicology in rats, and its performance in CT imaging in rabbits. RESULTS: BAY-576 is an extraordinarily stable chelate with a metal content of 42% (wt/wt) and with excellent water solubility. Formulations of 300 mg Hf/mL exhibited viscosity (3.3-3.6 mPa) and osmolality (860-985 mOsm/kg) in the range of nonionic x-ray agents. No relevant effects on erythrocytes, the coagulation, or complement system or on a panel of 87 potential biological targets were observed. The compound did not bind to plasma proteins of a number of species investigated. After intravenous injection in rats, it was excreted fast and mainly via the kidneys. Its pharmacokinetics was comparable to known extracellular contrast agents. A dose of 6000 mg Hf/kg, approximately 10 to 20 times the expected diagnostic dose, was well tolerated by rats with only moderate adverse effects. Computed tomography imaging in rabbits bearing a tumor in the liver demonstrated excellent image quality when compared with iopromide at the same contrast agent dose in angiography during the arterial phase. At 70% of the radiation dose, BAY-576 provided a contrast-to-noise ratio of the tumor, which was equivalent to iopromide at 100% radiation dose. CONCLUSIONS: The profile of BAY-576 indicates its potential as the first compound in a new class of noniodine x-ray contrast agents, which can contribute to the reduction of the radiation burden in contrast-enhanced CT imaging.

Glomerulonephritis-induced changes in kidney gene expression in rats.

We investigated a glomerulonephritis (GN) model in rats induced by nephrotoxic serum (NTS) which contains antibodies against the glomerular basement membrane (GBM). The anti-GBM GN model in rats is widely used since its biochemical and histopathological characteristics are similar to crescentic nephritis and Goodpasture's disease in humans (Pusey, 2003[2]). Male Wistar Kyoto (WKY) and Sprague-Dawley (SD) rats were dosed once with 1, 2.5 and 5 ml/kg nephrotoxic serum (NTS) or 1.5 and 5 ml/kg NTS, respectively. GN and tubular damage were observed histopathologically in all treated rats after 14 days. To obtain insight into molecular processes during GN pathogenesis, mRNA expression was investigated in WKY and SD kidneys using Affymetrix's GeneChip Rat genome 230_2.0 arrays (GSE64265). The immunopathological processes during GN are still not fully understood and likely involve both innate and adaptive immunity. In the present study, several hundred mRNAs were found deregulated, which functionally were mostly associated with inflammation and regeneration. The ß-chain of the major histocompatibility complex class II RT1.B (Rt1-Bb) and complement component 6 (C6) were identified as two mRNAs differentially expressed between WKY and SD rat strains which could be related to known different susceptibilities to NTS of different rat strains; both were increased in WKY and decreased in SD rats (Pavkovic et al., 2015 [1]). Increased Rt1-Bb expression in WKY rats could indicate a stronger and more persistent cellular reaction of the adaptive immune system in this strain, in line with findings indicating adaptive immune reactions during GN. The complement cascade is also known to be essential for GN development, especially terminal cascade products like C6.

Glomerulonephritis-Induced Changes in Urinary and Kidney MicroRNA Profiles in Rats.

MicroRNAs (miRNAs) regulate gene expression post-transcriptionally and thus are involved in various physiological and pathological states. Due to their stability in biofluids miRNAs have also been proposed as biomarkers (BMs) for tissue injury. We investigated the usefulness of urinary miRNAs for detection of site-specific renal damage in an antiglomerular basement membrane glomerulonephritis (GN) model in rats by comparing GN-induced urinary miRNAs profiles to traditional and newer protein BMs, and to proximal tubular injury-induced urinary miRNA profiles observed previously after cisplatin (Cp) treatment. Male Wistar Kyoto and Sprague Dawley rats were dosed once with 1, 2.5, and 5 ml/kg nephrotoxic serum (NTS) or 1.5 and 5 ml/kg NTS, respectively. GN and tubular damage were observed histopathologically in all treated rats after 14 days. Although serum creatinine and BUN were not changed, several protein BMs and 74 urinary miRNAs were found to be increased 8 and 14 days after NTS administration. Of these 74 miRNAs, 5 were identified as increased after NTS but not after Cp treatment. Using in situ hybridization two of them, miR-10 b and -100, were found to be localized in distal segments of the nephron, potentially reflecting the tubular injury in those regions. Furthermore, evaluation of both miRNA and mRNA expression in the kidney revealed possible miRNA-mRNA interactions mostly associated with fibrotic and transforming growth factor ß signaling. In conclusion, our investigations support the potential of urinary miRNAs as specific BMs for kidney injury, and suggest a role of miRNAs in pathological processes during GN in the kidney.

Urinary microRNA profiling for identification of biomarkers after cisplatin-induced kidney injury.

Extracellular microRNAs (miRNAs) have emerged as novel biomarkers (BMs) for various pathological states. To evaluate whether urinary miRNAs could serve as biomarkers for drug-induced kidney injury, we performed a nephrotoxicity study in rats with cisplatin (Cp), which is known to induce renal proximal tubular lesions in several species. Male Wistar rats were treated with a single dose of Cp (0, 1 and 3mg/kg) and urine was collected on days 3, 5, 8, 15 and 26 for measurement of several biomarkers and for RNA isolation. MiRNA profiling experiments with urine samples derived from the 3mg/kg Cp dosed animals, identified 136 miRNAs significantly increased in urine 3 and 5 days after Cp administration. 18 miRNAs with distinct time-dependent profiles were further analyzed using specific miRNA assays and absolute quantification. We observed >20-fold changes for 11 of these 18 miRNAs measured in profiling experiments, and confirmed their direction of change and time course profile by absolute quantification. Furthermore we found mechanistic links between several miRNAs and simultaneously measured mRNAs in the kidney after Cp administration. These were associated with pathways suggested to be involved in Cp-induced nephrotoxicity including a DNA damage response, apoptosis, and cell cycle regulation. Overall our results indicate that miRNAs measured in urine may serve as BMs for nephrotoxicity in rats.

3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions.

Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions.

Comparison of the MesoScale Discovery and Luminex multiplex platforms for measurement of urinary biomarkers in a cisplatin rat kidney injury model.

INTRODUCTION: In the past years several new urinary nephrotoxicity biomarkers have been qualified for use in preclinical studies by the FDA and EMA. Subsequently, kits have been developed to measure these urinary biomarkers on multiplex platforms such as the electro-chemiluminescent based immunoassay from MesoScale Discovery (MSD) and the bead-based immunoassay using Luminex xMAP technology (LMX). The aim of the present study was to compare the two multiplex platforms with respect to the capability of their qualified urinary biomarker panels to measure an increase of these biomarkers relative to histopathological changes in an animal model of nephrotoxicity. METHODS: For comparison of the two platforms we used urine samples from a study with the well-characterized nephrotoxin cisplatin (Cp) in male Wistar rats. The following five biomarkers were measured on both platforms: glutathione S-transferase α (αGST), clusterin (CLU), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL, a.k.a. lipocalin-2) and osteopontin (OPN). The measurements were compared with respect to both the fold increase observed for each biomarker and the absolute concentrations measured in relation to traditional endpoints for nephrotoxicity in clinical pathology and histopathology. RESULTS: The platform comparison revealed the expected increases of urinary biomarkers after Cp treatment with similar results at the fold change level enabling consistent detection of kidney injury. The comparison of the absolute concentrations of biomarkers measured in the two platforms showed differences, the extent of which was analyte-dependent. DISCUSSION: By comparison of two widely used multiplex platforms, MSD and LMX, for the detection of renal toxicity biomarkers in urine, we observed the expected increases of these biomarkers in response to Cp administration. Depending on the marker, significant differences could be found when comparing the absolute concentrations thus suggesting that baseline levels for each platform will have to be set separately.

Cyanine dyes as contrast agents for near-infrared imaging in vivo: acute tolerance, pharmacokinetics, and fluorescence imaging.

We compare pharmacokinetic, tolerance, and imaging properties of two near-IR contrast agents, indocyanine green (ICG) and 1,1(')-bis-(4-sulfobutyl) indotricarbocyanine-5,5(')-dicarboxylic acid diglucamide monosodium salt (SIDAG). ICG is a clinically approved imaging agent, and its derivative SIDAG is a more hydrophilic counterpart that has recently shown promising imaging properties in preclinical studies. The rather lipophilic ICG has a very short plasma half-life, thus limiting the time available to image body regions during its vascular circulation (e.g., the breast in optical mammography where scanning over several minutes is required). In order to change the physicochemical properties of the indotricarbocyanine dye backbone, several derivatives were synthesized with increasing hydrophilicity. The most hydrophilic dye SIDAG is selected for further biological characterization. The acute tolerance of SIDAG in mice is increased up to 60-fold compared to ICG. Contrary to ICG, the pharmacokinetic properties of SIDAG are shifted toward renal elimination, caused by the high hydrophilicity of the molecule. N-Nitrosomethylurea (NMU)-induced rat breast carcinomas are clearly demarcated, both immediately and 24 h after intravenous administration of SIDAG, whereas ICG shows a weak tumor contrast under the same conditions. Our findings demonstrate that SIDAG is a high potential contrast agent for optical imaging, which could increase the sensitivity for detection of inflamed regions and tumors.

The enhanced value of combining conventional and "omics" analyses in early assessment of drug-induced hepatobiliary injury.

The InnoMed PredTox consortium was formed to evaluate whether conventional preclinical safety assessment can be significantly enhanced by incorporation of molecular profiling ("omics") technologies. In short-term toxicological studies in rats, transcriptomics, proteomics and metabolomics data were collected and analyzed in relation to routine clinical chemistry and histopathology. Four of the sixteen hepato- and/or nephrotoxicants given to rats for 1, 3, or 14days at two dose levels induced similar histopathological effects. These were characterized by bile duct necrosis and hyperplasia and/or increased bilirubin and cholestasis, in addition to hepatocyte necrosis and regeneration, hepatocyte hypertrophy, and hepatic inflammation. Combined analysis of liver transcriptomics data from these studies revealed common gene expression changes which allowed the development of a potential sequence of events on a mechanistic level in accordance with classical endpoint observations. This included genes implicated in early stress responses, regenerative processes, inflammation with inflammatory cell immigration, fibrotic processes, and cholestasis encompassing deregulation of certain membrane transporters. Furthermore, a preliminary classification analysis using transcriptomics data suggested that prediction of cholestasis may be possible based on gene expression changes seen at earlier time-points. Targeted bile acid analysis, based on LC-MS metabonomics data demonstrating increased levels of conjugated or unconjugated bile acids in response to individual compounds, did not provide earlier detection of toxicity as compared to conventional parameters, but may allow distinction of different types of hepatobiliary toxicity. Overall, liver transcriptomics data delivered mechanistic and molecular details in addition to the classical endpoint observations which were further enhanced by targeted bile acid analysis using LC/MS metabonomics.

NMR and MS methods for metabonomics.

Metabonomics, also often referred to as "metabolomics" or "metabolic profiling," is the systematic profiling of metabolites in bio-fluids or tissues of organisms and their temporal changes. In the last decade, metabonomics has become increasingly popular in drug development, molecular medicine, and other biotechnology fields, since it profiles directly the phenotype and changes thereof in contrast to other "-omics" technologies. The increasing popularity of metabonomics has been possible only due to the enormous development in the technology and bioinformatics fields. In particular, the analytical technologies supporting metabonomics, i.e., NMR, LC-MS, UPLC-MS, and GC-MS have evolved into sensitive and highly reproducible platforms allowing the determination of hundreds of metabolites in parallel. This chapter describes the best practices of metabonomics as seen today. All important steps of metabolic profiling in drug development and molecular medicine are described in great detail, starting from sample preparation, to determining the measurement details of all analytical platforms, and finally, to discussing the corresponding specific steps of data analysis.

Evaluation of novel biomarkers of nephrotoxicity in two strains of rat treated with Cisplatin.

Cisplatin is an anticancer agent that induces renal proximal tubule lesions in many species. Studies were conducted in Sprague-Dawley and Han-Wistar rats to evaluate the utility of novel preclinical biomarkers of nephrotoxicity for renal lesions caused by this compound. Groups of 10 males of each strain were given a single intraperitoneal injection of 0.3, 1, or 3 mg/kg cisplatin and were sacrificed on days 2, 3, and 5. The novel biomarkers α-glutathione-S-transferase (α-GST) (for proximal tubular injury), µ-glutathione-S-transferase (µ-GST) (for distal tubular injury), clusterin (for general kidney injury), and renal papillary antigen-1 (RPA-1) (for collecting duct injury) were measured in urine by enzyme immunoassay. Histologically, degeneration and necrosis of the S3 segment of the renal proximal tubule were observed on day 2 (Han-Wistar) and days 3 and 5 (both strains) at 1 and 3 mg/kg. Results showed that in both strains of rats, urinary α-GST and clusterin can be detected in urine soon after injury, are more sensitive than BUN and serum creatinine, and therefore are usable as noninvasive biomarkers of proximal tubule injury. Changes in both µ-GST or RPA-1 were considered to represent secondary minor effects of proximal tubular injury on distal segments of the nephron.