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Unlabelled: Hepatocellular carcinoma (HCC) occurs predominantly in patients with liver cirrhosis. Here we show an innovative RNA-based targeted approach to enhance endogenous albumin production while reducing liver tumor burden. We designed short-activating RNAs (saRNA) to enhance expression of C/EBPα (CCAAT/enhancer-binding protein-α), a transcriptional regulator and activator of albumin gene expression. Increased levels of both C/EBPα and albumin mRNA in addition to a 3-fold increase in albumin secretion and 50% decrease in cell proliferation was observed in C/EBPα-saRNA transfected HepG2 cells. Intravenous injection of C/EBPα-saRNA in a cirrhotic rat model with multifocal liver tumors increased circulating serum albumin by over 30%, showing evidence of improved liver function. Tumor burden decreased by 80% (P = 0.003) with a 40% reduction in a marker of preneoplastic transformation. Since C/EBPα has known antiproliferative activities by way of retinoblastoma, p21, and cyclins, we used messenger RNA (mRNA) expression liver cancer-specific microarray in C/EBPα-saRNA-transfected HepG2 cells to confirm down-regulation of genes strongly enriched for negative regulation of apoptosis, angiogenesis, and metastasis. Up-regulated genes were enriched for tumor suppressors and positive regulators of cell differentiation. A quantitative polymerase chain reaction (PCR) and western blot analysis of C/EBPα-saRNA-transfected cells suggested that in addition to the known antiproliferative targets of C/EBPα, we also observed suppression of interleukin (IL)6R, c-Myc, and reduced STAT3 phosphorylation., Conclusion: A novel injectable saRNA-oligonucleotide that enhances C/EBPα expression successfully reduces tumor burden and simultaneously improves liver function in a clinically relevant liver cirrhosis/HCC model., (© 2013 by the American Association for the Study of Liver Diseases.)

During the past few decades, the development and use of individually ventilated cages (IVC), which are now commercially available for housing laboratory mice and rats, have increased. Because limited information is available regarding the influence of caging systems on the growth of rats, the present study assessed body weight and food and water consumption in growing male rats that were housed in IVC and open-top cages (OTC). We allocated 21-d-old male Wistar outbred rats (HsdOla:WI; n = 24) into 2 groups, which then were housed in pairs in IVC (n = 12) and OTC (n = 12). After an 8-d acclimatization period, body weight and food and water consumption were assessed every 3 d until the rats were 94 d old. There were no significant differences between the body weights of rats housed in IVC compared with OTC over the 65-d observation period. Food and water consumption were greater in rats housed in OTC compared with IVC, becoming significantly different when the rats were 50 and 53 d old, respectively. In conclusion, IVC and OTC housing conditions influenced food and water intakes but not body weight in growing male rats. Further research is needed to clarify the exact basis for these changes in food and water consumption.

The incretin hormone Glucose-dependent Insulinotropic Polypeptide GIP1-42 (approximately 5 kDa), is released postprandially, and rapidly degraded by Dipeptidyl Peptidase IV (DP-IV) to yield the inactive GIP3-42. Methods for the quantification of the pair of GIP peptides include combinations of immunoassays; however, mass spectrometry based approaches can offer the improved selectivity required for the distinction between the active and inactive forms. In this study, we report an LC/ESI-MS/MS approach for the simultaneous absolute quantification of GIP1-42 and GIP3-42 via the corresponding surrogate proteolytic peptide fragments, GIP1-16 and GIP3-16. These surrogate peptides afford approximately 250-fold improvement in lower limits of quantification (LLOQ) compared to the precursor proteins. The LLOQ of the reported method was 5 ng/mL (5-1000 ng/mL) for GIP1-42 and 10 ng/mL (10-1000 ng/mL) for GIP3-42, using 100 microL of mouse plasma. This is the first reported study in which the GIP1-42 and GIP3-42 polypeptides are quantified simultaneously with LC/ESI-MS/MS via their tryptic surrogate peptides. The approach is suitable for both preclinical and clinical pharmacokinetic studies due to the low volume required for the analysis. The described methodology was applied to a pharmacokinetic study, in which enhanced stability of exogenously administered GIP1-42 was demonstrated in mice treated with a DP-IV inhibitor.

The study of pharmacologically active peptides is central for the understanding of cancer and the development of novel therapeutic approaches. In this context, both qualitative and quantitative determination of bioactive peptides in biological fluids/tissues and their effect on endogenous factors (e.g. hormones) are of great importance. A mass spectrometry-based approach was developed and applied towards the measurement of leuprolide, a peptide drug for the treatment of prostate cancer, in mouse plasma. High-pressure liquid chromatography coupled to a hybrid quadrupole linear ion trap (QqLIT) mass spectrometer, a platform that combines the benefits of triple QqLIT instruments, was employed for the study. Using the described methodology, we established that picomolar concentrations of leuprolide could be measured in mouse plasma (limit of quantification of 0.1 ng/ml). In order to optimize pharmacokinetic properties of analogs of leuprolide, a facile in vivo mouse model was developed and leuprolide concentrations were determined in mouse plasma following intraperitoneal administration. In the same animal model, we demonstrated the versatility of the described MS-based approach by the determination of plasma concentrations of testosterone, an established biomarker for the treatment of prostate cancer. Following dosing with leuprolide, circulating testosterone was increased significantly in comparison to vehicle-treated mice. Finally, in vitro metabolism of leuprolide was evaluated by incubation of leuprolide with mouse kidney membranes, followed by identification of major metabolites by MS. Such studies provide the framework for future evaluation of novel leuprolide analogs with potential therapeutic advantages.