Abstract Poly (ADP-ribose) polymerase (PARP) inhibitors exert their effect intracellularly within tumor, thus sufficient tumor penetration is essential for a pharmacological response. Preclinical mouse xenograft data show a 3.3-fold higher tumor versus plasma exposure of niraparib, while for olaparib tumor exposure was less than plasma. This study aimed to build a physiologically-based pharmacokinetic (PBPK) model extended with a tissue composition-based permeability-limited tumor model to: (a) gain a mechanistic understanding of the differences in tumor exposure of niraparib and olaparib; and (b) to predict clinical tumor exposure in ovarian cancer patients at clinically relevant dosing regimens. A permeability-limited tumor model was developed that integrates data on tumor composition and drug physicochemical properties analogous to the established permeability-limited organ model available for the liver in the Simcyp Simulator [1,2]. The model assumes that unbound unionized drug is in equilibrium between the vascular and interstitial compartments and movement of the drug between the interstitial and intracellular space is via passive permeability. Total tumor concentration is dependent on passive permeability of the drug, drug binding to PARP and nonspecific binding to neutral lipids, neutral phospholipids, and acidic phospholipids in the intracellular space, albumin in the interstitial and pH of the tumor interstitial and intracellular spaces. Clinical and preclinical tumor physiological parameters such as volume, blood flow, and tissue composition are defined using published data. The model was developed using the Simcyp Simulator V17.1 and R [3]. Consistent with preclinical data, the model predicts a 5-7-fold higher tumor exposure relative to plasma, as measured by the AUC tumor to plasma ratio of niraparib compared with olaparib. Significant binding to acidic phospholipids contributes to the increased tumor exposure to niraparib, a basic drug, a mechanism that is not relevant to the neutral drug olaparib. Ongoing work aims to extrapolate the model to predict the clinical tumor concentration of olaparib and niraparib in ovarian cancer patients and to investigate the sensitivity of the model to key tumor attributes, including blood flow and interstitial pH that may contribute to variability in tumor drug exposure. A similar modeling approach may be used to predict the tumor exposure of other small molecule anticancer drugs from their plasma concentration and physicochemical properties in different types of solid tumor. References Jamei M et al, Clin Pharmacokinet. 2014;53:73-87 Poulin P et al, J Pharm Sci. 2015;104:1508-21 R Core Team (2018). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/. Citation Format: Rachel H. Rose, Kaiming Sun, Linzhong Li, Keyur Gada, Jing Yu Wang, Yongchang Qiu. Predicting concentration of PARP inhibitors in human tumor tissue using PBPK modeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2952.
Aims: Heparan sulfate (HS) accumulates in the central nervous system in mucopolysaccharidosis III type A (MPS IIIA). A validated LC–MS/MS assay was developed to measure HS in human cerebrospinal fluid (CSF). Methods & results: HS was extracted and digested and the resultant disaccharides were derivatized with a novel label, 4-butylaniline, enabling isoform separation and isotope-tagged analog introduction as an internal standard for LC–MS/MS. The assay has a LLOQ for disaccharides of 0.1 μM, ±20% accuracy and ≤20% precision. CSF samples from patients with MPS IIIA showed elevated HS levels (mean 4.9 μM) compared with negative controls (0.37 μM). Conclusion: This assay detected elevated HS levels in the CSF of patients with MPS IIIA and provides a method to assess experimental therapies.
The functional and structural alterations of vascular endothelium contribute to the initiation, progression, and complications of atherosclerotic plaque formation, but limited information is known about the molecular composition and pathways underlying pathological changes during atherosclerosis. We have developed an affinity proteomic strategy for in situ isolation and differential mapping of vascular endothelial proteins in normal and atherosclerotic aorta tissues. The selective labeling was carried out by perfusion of the blood vessels with an active biotin reagent for covalent modification of accessible vascular endothelial proteins. The biotinylated proteins were then enriched by streptavidin affinity chromatography, separated by SDS-PAGE, and subsequently characterized by LC-MS/MS. The described procedure led to the identification of 454 distinct proteins in normal and atherosclerotic aorta tissues. A majority of the proteins are plasma membrane associated and extracellular matrix proteins, and 81 showed altered expressions in atherosclerotic aorta tissue. The differentially expressed proteins are involved in immune and inflammatory responses, cell adhesion, and lipid metabolism. The method provides a new avenue for investigating the endothelial dysfunction and development of atherosclerosis.
The 2018 12th Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9–13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in 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 bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 2 (hybrid LBA/LCMS for biotherapeutics and regulatory agencies' inputs) are published in volume 10 of Bioanalysis, issues 22 and 23 (2018), respectively.
Aim: To provide more efficient and timely immunogenicity testing service to support routine patient care, the original complex testing algorithm for evaluation of anti-velaglucerase alfa antibodies has been simplified and individual methods (screen, confirm, titer, neutralizing antibody [NAb] and IgE) have been redeveloped/optimized and validated. Results: To compare the performance of different methods, 50 velaglucerase alfa-treated patient samples were analyzed using both old and new methods for the presence of antidrug antibodies (ADAs) and 31 ADA-positive samples were analyzed for neutralizing capacity. The ADA and NAb statuses are almost identical from both methods and both ADA and NAb titer results are highly correlated with a Spearman's correlation of 0.96 and 0.86, respectively. Conclusion: The original and new testing methods can be considered interchangeable for the measurement of total and neutralizing anti-velaglucerase alfa antibodies.
Lymphotoxin-β receptor (LTβR) is a member of tumor necrosis factor receptor family and plays essential roles in the embryonic development and organization of secondary lymphoid tissues. It binds two types of tumor necrosis factor family cytokines, heterotrimer LTα1β2 and homotrimer LIGHT, and activates multiple signaling pathways including transcriptional factor NFκB, c-Jun N-terminal kinase, and cell death. However, the molecular mechanism of the activation of these signaling pathways by LTβR is not clear. Because there is no enzymatic activity associated with the receptor itself, the signal transduction of LTβR is mediated by cytoplasmic proteins recruited to receptors. To identify these proteins, we took a proteomic approach. The endogenous LIGHT·LTβR complex was affinity-purified from U937 cells, and proteins associated with the complex were identified by mass spectrometry. Four of five proteins identified, TRAF2, TRAF3, cIAP1, and Smac, are reported here. Their association with LTβR was further confirmed by coimmunoprecipitation in U937 cells and HEK293 cells. The presence of cIAP1 and Smac in LIGHT·LTβR complex revealed a novel mechanism of LIGHT·LTβR-induced apoptosis.
Aim: The study aimed to develop an LC–MS/MS assay to measure dermatan sulfate (DS) in human cerebrospinal fluid (CSF). Methods & results: DS was quantified by ion pairing LC–MS/MS analysis of the major disaccharides derived from chondroitinase B digestion. Artificial CSF was utilized as a surrogate for calibration curve preparation. The assay was fully validated, with a linear range of 20.0–4000 ng/ml, accuracy within ±20%, and precision of ≤20%. CSF samples from mucopolysaccharidoses (MPS) II patients showed an average of 11-fold increase in DS levels compared with controls. Conclusion: The described assay is capable of differentiating DS levels in the CSF of MPS II patients from controls and can be used to monitor disease progression and therapeutic responses.
The prevention of adventitious agent contamination is a top priority throughout the entire biopharmaceutical production process. For example, although viral contamination of cell banks or cell cultures is rare, it can result in serious consequences (e.g., shutdown and decontamination of manufacturing facilities). To ensure virus free production, numerous in vivo and in vitro adventitious agent assays and biophysical characterizations such as electron microscopy are conducted on cell banks, raw materials, process materials, and drug substances throughout the manufacturing process. Molecular assays such as PCR and other nucleotide-based techniques are also routinely used for screening and identification of any viral agents. However, modern techniques in protein identification of complex protein mixtures have not yet been effectively integrated throughout the industry into current viral testing strategies. Here, we report the identification and quantitation of Vesivirus 2117 particles in bioreactor fluid from infected Chinese hamster ovary cell cultures by global protein sequencing using mass spectrometry in combination with multi-dimensional liquid-chromatography. Following mass spectrometric data acquisition and rigorous data analysis, six virus specific peptides were identified. These peptides were fragments of two structural proteins, capsid protein pre-cursor (four unique peptides) and small structural protein (two unique peptides), from the same species: Vesivirus 2117. Using stable heavy isotope-labeled peptides as internal standards, we also determined the absolute concentration of Vesivirus particles in the bioreactor fluid and the ratio of two capsid proteins (VP1:VP2) in the particles as approximately 9:1. The positive identification of Vesivirus 2117 was subsequently confirmed by RT-PCR.
