Brain natriuretic peptide (BNP) and N-terminal proBNP (NTproBNP) are well established in the clinic as biomarkers of heart failure. BNP hormone and the inactive NTproBNP are predominantly secreted in the ventricles of the heart in response to pressure overload and, consequently, are being investigated as markers of drug-induced cardiac hypertrophy in rat to support drug development. In the work presented here, an immunoaffinity-based LC/MS/MS assay was developed and validated to measure a selective tryptic fragment of NTproBNP in rat serum. The assay covers the range of 13-329 pg/mL of the tryptic fragment LLELIR, corresponding to 0.1-2.5 ng/mL intact NTproBNP. A stable isotope-labeled version of NTproBNP containing the tryptic fragment LLELI[13C615N1]R was prepared by solid-phase peptide synthesis and was used as an internal standard to minimize assay variability. Due to endogenous NTproBNP present in rat serum, human serum was used as the control matrix, and parallelism between rat and human serum was established by standard addition. Assay accuracy (% RE) and precision (% CV) were measured at three concentrations on each of 4 days and did not exceed 4.2 and 14.5%, respectively. Additionally, study data are presented from the application of this assay in which rats demonstrated a significant increase in NTproBNP serum concentration following administration of an agent known to induce cardiac hypertrophy. In this study, the relationship between serum NTproBNP and cardiac hypertrophy was corroborated by increases in heart weight and magnetic resonance imaging of the test subjects' left ventricle. To our knowledge, this represents the first reported assay for NTproBNP in preclinical species for the assessment of cardiac hypertrophy.

The pro-inflammatory cytokines, Tumor Necrosis Factor-alpha (TNFα) and Interleukin-1 (IL-1) mediate the innate immune response. Dysregulation of the innate immune response contributes to the pathogenesis of cancer, arthritis, and congestive heart failure. TNFα-and IL-1-induced changes in gene expression are mediated by similar transcription factors; however, TNFα and IL-1 receptor knock-out mice differ in their sensitivities to a known initiator (lipopolysaccharide, LPS) of the innate immune response. The contrasting responses to LPS indicate that TNFα and IL-1 regulate different processes. A large-scale proteomic analysis of TNFα-and IL-1-induced responses was undertaken to identify processes uniquely regulated by TNFα and IL-1. When combined with genomic studies, our results indicate that TNFα, but not IL-1, mediates cell cycle arrest.

Current drug discovery involves a highly iterative process pertaining to three core disciplines: biology, chemistry, and drug disposition. For most pharmaceutical companies the path to a drug candidate comprises similar stages: target identification, biological screening, lead generation, lead optimization, and candidate selection. Over the past decade, the overall efficiency of drug discovery has been greatly improved by a single instrumental technique, liquid chromatography/mass spectrometry (LC/MS). Transformed by the commercial introduction of the atmospheric pressure ionization interface in the mid-1990s, LC/MS has expanded into almost every area of drug discovery. In many cases, drug discovery workflow has been changed owing to vastly improved efficiency. This review examines recent trends for these three core disciplines and presents seminal examples where LC/MS has altered the current approach to drug discovery.

No abstract

No abstract