Publications by authors named "Mark Cancilla"

Embedded within the field of drug metabolism and pharmacokinetics (DMPK), biotransformation is a discipline that studies the origins, disposition, and structural identity of metabolites to provide a comprehensive safety assessment, including the assessment of exposure coverage in toxicological species. Spanning discovery and development, metabolite identification (metID) scientists employ various strategies and tools to address stage-specific questions aimed at guiding the maturation of early chemical matter into drug candidates. During this process, the identity of major (and minor) circulating human metabolites is ascertained to comply with the regulatory requirements such as the Metabolites in Safety Testing (MIST) guidance.

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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.

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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.

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Over the past three decades, mass spectrometry imaging (MSI) has emerged as a valuable tool for the spatial localization of drugs and metabolites directly from tissue surfaces without the need for labels. MSI offers molecular specificity, making it increasingly popular in the pharmaceutical industry compared to conventional imaging techniques like quantitative whole-body autoradiography (QWBA) and immunohistochemistry, which are unable to distinguish parent drugs from metabolites. Across the industry, there has been a consistent uptake in the utilization of MSI to investigate drug and metabolite distribution patterns, and the integration of MSI with omics technologies in preclinical investigations.

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The discovery of peptide therapeutics represents a fast-growing segment of pharmaceutical research. During the early discovery process, a large number of peptide candidates needs to be rapidly screened for metabolic stability in relevant biological matrices. In most cases, peptide stability assays are quantified using LC-MS/MS, which may take hours to analyze 384 samples and generates liters of solvent waste.

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Glucuronidation is a common phase II metabolic process for drugs and xenobiotics which increases their solubility for excretion. Acyl glucuronides (glucuronides of carboxylic acids) present concerns as they have been implicated in gastrointestinal toxicity and hepatic failure. Despite the substantial success in the bulk analysis of these species, previous attempts using traditional mass spectrometry imaging (MSI) techniques have completely or partially failed and therefore little is known about their localization in tissues.

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Acyl glucuronide (AG) metabolites of carboxylic acid-containing drugs and products of their transformations have long been implicated in drug-induced liver injury (DILI). To inform on the DILI risk arising from AG reactive intermediates, a comprehensive mechanistic study of enzyme-independent AG rearrangements using nuclear magnetic resonance (NMR) and density functional theory (DFT) was undertaken. NMR spectroscopy was utilized for structure elucidation and kinetics measurements of nine rearrangement and hydrolysis products of 1β-O-acyl glucuronide of ibufenac.

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dropletProbe mass spectrometry is a novel technique for molecular characterization of surfaces. It can be used for rapid ex vivo analysis of therapeutics from thin animal tissue sections and has been shown to improve understanding of a drug's absorption, distribution, metabolism and excretion (ADME) properties. Here, we describe the tissue distribution analysis of diclofenac from a dosed whole-body mouse thin tissue section using a dropletProbe mass spectrometry system.

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Bruton's tyrosine kinase (BTK) is an essential node on the BCR signaling in B cells, which are clinically validated to play a critical role in B-cell lymphomas and various auto-immune diseases such as Multiple Sclerosis (MS), Pemphigus, and rheumatoid arthritis (RA). Although non-selective irreversible BTK inhibitors have been approved for oncology, due to the emergence of drug resistance in B-cell lymphoma associated with covalent inhibitor, there an unmet medical need to identify reversible, selective, potent BTK inhibitor as viable therapeutics for patients. Herein, we describe the identification of Hits and subsequence optimization to improve the physicochemical properties, potency and kinome selectivity leading to the discovery of a novel class of BTK inhibitors.

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Peptides have become a fast-growing segment of the pharmaceutical industry over the past few decades. It is essential to develop cutting edge analytical techniques to support the discovery and development of peptide therapeutics, especially to examine their absorption, distribution, metabolism and excretion (ADME) properties. Herein, we utilized two label-free mass spectrometry (MS) based techniques to investigate representative challenges in developing therapeutic peptides, such as tissue distribution, metabolic stability and clearance.

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dropletProbe mass spectrometry (MS) is an emerging tool for the rapid ex vivo analysis of drugs in tissues and whole-body sections. Its use has been demonstrated to better understand a drug's absorption, distribution, metabolism, and excretion (ADME) properties. To further optimize the overall utility of this technique, it is important to characterize and understand the various tissue matrix effects and extraction solvents on the overall performance of dropletProbe MS analyses.

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As metabolism impacts the efficacy and safety of therapeutic peptides and proteins (TPPs), understanding of the metabolic fate of TPPs is critical for their preclinical and clinical development. Despite the continued increase of new TPPs entering clinical trials, the metabolite identification (MetID) of these emerging modalities remains challenging. In the present study, we report an analytical workflow for MetID of TPPs.

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Rationale: Liquid chromatography/mass spectrometry is an essential tool for efficient and reliable quantitative and qualitative analysis and underpins much of contemporary drug metabolism and pharmacokinetics. Data-independent acquisition methods such as MS have reduced the potential to miss metabolites, but do not formally generate quadrupole-resolved product ion spectra. The addition of ion mobility separation to these approaches, for example, in High-Definition MS (HDMS ) has the potential to reduce the time needed to set up an experiment and maximize the chance that all metabolites present can be resolved and characterized.

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MK-8666, a selective GPR40 agonist developed for the treatment of type 2 diabetes mellitus, was discontinued in phase I clinical trials due to liver safety concerns. To address whether chemically reactive metabolites played a causative role in the observed drug induced liver injury (DILI), we characterized the metabolism, covalent binding to proteins, and amino acid targets of MK-8666 in rat and human hepatocytes or cofactor-fortified liver microsomes. MK-8666 was primarily metabolized to an acyl glucuronide in hepatocytes of both species and a taurine conjugate in rat hepatocytes.

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Glucuronidation, a common phase II biotransformation reaction, is one of the major and metabolism pathways of xenobiotics. In this process, glucuronic acid is conjugated to a drug or a drug metabolite via a carboxylic acid, a hydroxy, or an amino group to form acyl-, -, and/or -glucuronide metabolites, respectively. This process is traditionally thought to be a detoxification pathway.

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Since the approval of ibrutinib for the treatment of B-cell malignancies in 2012, numerous clinical trials have been reported using covalent inhibitors to target Bruton's tyrosine kinase (BTK) for oncology indications. However, a formidable challenge for the pharmaceutical industry has been the identification of reversible, selective, potent molecules for inhibition of BTK. Herein, we report application of Tethering-fragment-based screens to identify low molecular weight fragments which were further optimized to improve on-target potency and ADME properties leading to the discovery of reversible, selective, potent BTK inhibitors suitable for pre-clinical proof-of-concept studies.

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Drug combinations can improve the control of diseases involving redundant and highly regulated pathways. Validating a multi-target therapy early in drug development remains difficult. Small interfering RNAs (siRNAs) are routinely used to selectively silence a target of interest.

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Glucocorticoids (GCs) are excellent anti-inflammatory drugs but are dose-limited by on-target toxicity. We sought to solve this problem by delivering GCs to immune cells with antibody-drug conjugates (ADCs) using antibodies containing site-specific incorporation of a non-natural amino acid, novel linker chemistry for in vitro and in vivo stability, and existing and novel glucocorticoid receptor (GR) agonists as payloads. We directed fluticasone propionate to human antigen-presenting immune cells to afford GR activation that was dependent on the targeted antigen.

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The 2017 11th Workshop on Recent Issues in Bioanalysis (11th WRIB) took place in Los Angeles/Universal City, California on 3-7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecule analysis involving LCMS, hybrid ligand binding assay (LBA)/LCMS and LBA approaches.

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The 2017 11th Workshop on Recent Issues in Bioanalysis (11th WRIB) took place in Los Angeles/Universal City, California from 3 April 2017 to 7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - A Full Immersion Week of Bioanalysis, Biomarkers and Immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecule analysis involving LCMS, hybrid LBA/LCMS and ligand-binding assay (LBA) approaches.

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Small interfering RNA (siRNA)-mediated silencing requires siRNA loading into the RNA-induced silencing complex (RISC). Presence of 5'-phosphate (5'-P) is reported to be critical for efficient RISC loading of the antisense strand (AS) by anchoring it to the mid-domain of the Argonaute2 (Ago2) protein. Phosphorylation of exogenous duplex siRNAs is thought to be accomplished by cytosolic Clp1 kinase.

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As part of an effort to examine the utility of antibody-drug conjugates (ADCs) beyond oncology indications, a novel pyrophosphate ester linker was discovered to enable the targeted delivery of glucocorticoids. As small molecules, these highly soluble phosphate ester drug linkers were found to have ideal orthogonal properties: robust plasma stability coupled with rapid release of payload in a lysosomal environment. Building upon these findings, site-specific ADCs were made between this drug linker combination and an antibody against human CD70, a receptor specifically expressed in immune cells but also found aberrantly expressed in multiple human carcinomas.

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Two sets of synthetic 21-23mer oligonucleotides with various types of 2'-position modifications have been studied with tandem mass spectrometry using ion trap collision-induced dissociation (IT-CID) and negative electron transfer (NET)-CID. A systematic study has been conducted to define the limitations of IT-CID in sequencing such 2'-chemically modified oligonucleotides. We found that IT-CID is sufficient in characterizing oligonucleotide sequences that do not contain DNA residues, where high sequence coverage can be achieved by performing IT-CID on multiple charge states.

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We report the use of a fragment-based lead discovery method, Tethering with extenders, to discover a pyridinone fragment that binds in an adaptive site of the protein PDK1. With subsequent medicinal chemistry, this led to the discovery of a potent and highly selective inhibitor of PDK1, which binds in the 'DFG-out' conformation.

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Deeper knowledge of pharmacokinetic and pharmacodynamic (PK/PD) concepts for RNA therapeutics is important to streamline the drug development process and for rigorous selection of best performing drug candidates. Here we characterized the PK/PD relationship for small interfering RNAs (siRNAs) targeting luciferase by examining siRNA concentration in plasma and liver, the temporal RNA-induced silencing complex binding profiles, mRNA reduction, and protein inhibition measured by noninvasive bioluminescent imaging. A dose-dependent and time-related decrease in bioluminescence was detected over 25 days after a single treatment of a lipid nanoparticle-formulated siRNA targeting luciferase messenger RNA.

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