Leveraging High-Resolution Ion Mobility-Mass Spectrometry for Cyclic Peptide Soft Spot Identification.

Cyclic peptides are an important class of molecules that gained significant attention in the field of drug discovery due to their unique pharmacological characteristics and enhanced proteolytic stability. Yet, gastrointestinal degradation remains a major hurdle in the discovery of orally bioavailable cyclic peptides. Soft spot identification (SSID) of the regions in the cyclic peptide sequence susceptible to amide hydrolysis by proteases is used in the discovery stage to guide medicinal chemistry design.

Rapid and Definitive Identification of Cyclic Peptide Soft Spots by Isotope-Labeled Reductive Dimethylation and Mass Spectrometry Fragmentation.

Cyclic peptides are an emerging therapeutic modality over the past few decades. To identify drug candidates with sufficient proteolytic stability for oral administration, it is critical to pinpoint the amide bond hydrolysis sites, or soft spots, to better understand their metabolism and provide guidance on further structure optimization. However, the unambiguous characterization of cyclic peptide soft spots remains a significant challenge during early stage discovery studies, as amide bond hydrolysis forms a linearized isobaric sequence with the addition of a water molecule, regardless of the amide hydrolysis location.

Universal Accessible Biomarkers of Drug-Induced Tissue Injury and Systemic Inflammation in Rat: Performance Assessment of TIMP-1, A2M, AGP, NGAL, and Albumin.

The ability to monitor for general drug-induced tissue injury (DITI) or systemic inflammation in any tissue using blood-based accessible biomarkers would provide a valuable tool in early exploratory animal studies to understand potential drug liabilities. Here we describe the evaluation of 4 biomarkers of tissue remodeling and inflammation (α2-macroglobulin [A2M], α1-acid glycoprotein [AGP], neutrophil gelatinase-associated lipocalin [NGAL], and tissue inhibitor of metalloproteinases [TIMP-1]) as well as the traditional serum parameter albumin as potential blood-based biomarkers of DITI and systemic inflammatory response (SIR). Biomarker performance was assessed in 51 short-term rat in vivo studies with various end-organ toxicities or SIR and receiver operating characteristic curves were generated to compare relative performances.

The application of cytokeratin-18 as a biomarker for drug-induced liver injury.

Drug-induced liver injury (DILI) is a frequent and dangerous adverse effect faced during preclinical and clinical drug therapy. DILI is a leading cause of candidate drug attrition, withdrawal and in clinic, is the primary cause of acute liver failure. Traditional diagnostic markers for DILI include alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP).

Use of a Bile Salt Export Pump Knockdown Rat Susceptibility Model to Interrogate Mechanism of Drug-Induced Liver Toxicity.

Inhibition of the bile salt export pump (BSEP) may be associated with clinical drug-induced liver injury, but is poorly predicted by preclinical animal models. Here we present the development of a novel rat model using siRNA knockdown (KD) of Bsep that displayed differentially enhanced hepatotoxicity to 8 Bsep inhibitors and not to 3 Bsep noninhibitors when administered at maximally tolerated doses for 7 days. Bsep KD alone resulted in 3- and 4.

Vascular Imaging of Matrix Metalloproteinase Activity as an Informative Preclinical Biomarker of Drug-induced Vascular Injury.

Lack of biomarkers specific to and either predictive or diagnostic of drug-induced vascular injury (DIVI) continues to be a major obstacle during drug development. Biomarkers derived from physiologic responses to vessel injury, such as inflammation and vascular remodeling, could make good candidates; however, they characteristically lack specificity for vasculature. We evaluated whether vascular remodeling-associated protease activity, as well as changes to vessel permeability resulting from DIVI, could be visualized ex vivo in affected vessels, thereby allowing for visual monitoring of the pathology to address specificity.

Optical imaging biomarkers of drug-induced vascular injury.

Drug-induced vascular injury (DIVI), defined as arterial medial degeneration/necrosis usually associated with perivascular inflammation, is frequently observed in the mesenteric arteries of rats but the relevance to humans remains a hurdle for drug development. Here, we describe the evaluation of commercially available optical imaging biomarkers using a rat DIVI model. Male Sprague Dawley rats were administered 10 mg/kg/day of a proprietary soluble guanylate cyclase activator (sGCa).

Nonclinical safety biomarkers of drug-induced vascular injury: current status and blueprint for the future.

Better biomarkers are needed to identify, characterize, and/or monitor drug-induced vascular injury (DIVI) in nonclinical species and patients. The Predictive Safety Testing Consortium (PSTC), a precompetitive collaboration of pharmaceutical companies and the U.S.

A performance evaluation of three drug-induced liver injury biomarkers in the rat: alpha-glutathione S-transferase, arginase 1, and 4-hydroxyphenyl-pyruvate dioxygenase.

Alanine aminotransferase (ALT) activity is the most frequently relied upon reference standard for monitoring liver injury in humans and nonclinical species. However, limitations of ALT include a lack of specificity for diagnosing liver injury (e.g.

Expression and activities of several drug-metabolizing enzymes in LLC-PK1 cells.

LLC-PK1 cells are frequently used in toxicology research, but little information is available concerning the capacity of these cells to metabolize xenobiotics. We examined the expression and activities of cytochromes P450 (P450) 1A1/1A2 (CYP 1A1/1A2), 2E1 (CYP 2E1), flavin monooxygenase (FMO), 5-lipoxygenase (5-LO) and prostaglandin H synthase (PHS)-associated cyclooxygenase-1 (COX-1). We prepared S9 fractions from LLC-PK1 cells, rat liver, and rat kidney, and measured enzyme activities using ethoxyresorufin O-deethylation (EROD) for CYP 1A1/1A2 and ethoxycoumarin O-deethylation (ECOD) for CYP 2E1, benzydamine N-oxidation (BNO) for FMO, leukotriene B(4) (LTB(4)) formation for 5-LO, and thromboxane B(2) (TXB(2)) formation for COX-1 activities.