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A biomimetic assay platform for the interrogation of antigen-dependent anti-tumor T-cell function
No full textOvercoming tumor-mediated immunosuppression and enhancing cytotoxic T-cell activity within the tumor microenvironment are two central goals of immuno-oncology (IO) drug discovery initiatives. However, exploratory assays involving immune components are often plagued by low-throughput and poor clinical relevance. Here we present a novel ultra-high-content assay platform for interrogating T-cell-mediated killing of 3D multicellular tumor spheroids. Employing this assay platform in a chemical genomics screen of 1,800 annotated compounds enabled the identification of novel small molecule perturbagens capable of enhancing cytotoxic CD8+ T-cell activity in an antigen-dependent manner. Specifically, cyclin-dependent kinase (CDK) and bromodomain (BRD) protein inhibitors were shown to significantly augment anti-tumor T-cell function by increasing cytolytic granule and type II interferon expression. The described biotechnology screening platform yields multi-parametric, clinically-relevant data and can be employed kinetically for the discovery of novel IO therapeutic agents
Use of PBPK Modeling for Predicting Drug-Food Interactions: Successful prediction using a middle-out approach
No full textOver the last 10 years, 40% of approved oral drugs exhibited a significant effect of food on pharmacokinetics (PK) and currently the only method to characterize the effect of food on drug absorption which is recognized by the authorities, is to conduct a clinical evaluation. Within the pharmaceutical industry, there is a significant effort to predict the mechanism and clinical relevance of a food effect. Physiologically-based pharmacokinetic (PBPK) models combining both drug-specific and physiology-specific data have been used to interpret the effect of food on absorption and the underlying mechanisms. This manuscript provides detailed descriptions of how a middle-out modeling approach can be used to predict the magnitude and direction of food effect for three compounds: nefazodone, furosemide and aprepitant. For nefazodone, a mechanistic clearance for gut and liver was added, for furosemide, an absorption window was introduced and for aprepitant, the solubility, absorption scaling factors and volume needed adjustment. In all cases, these adjustments were supported by literature data and modelers judgement on the factors limiting absorption. Through this modelling, some differences in how software packages handle the data or make assumptions in default system parameters led to differences in the predicted food effect which is also presented for the three examples
FGF21 Normalizes Plasma Glucose in Mouse Models of Type 1 Diabetes and Insulin Receptor Dysfunction
No full textFibroblast growth factor 21 (FGF21) is a member of the fibroblast growth factor (FGF) family of proteins. The biological activity of FGF21 was first shown to induce insulin independent glucose uptake in adipocytes through the GLUT1 transporter. Subsequently it was shown to have effects on the liver to increase fatty acid oxidation. FGF21 treatment provides beneficial metabolic effects in both animal models and patients with obesity, type 2 diabetes mellitus (T2D) and/or fatty liver disease. In this paper, we revisited the original finding and found that the insulin independent glucose uptake in adipocytes is preserved in the presence of an insulin receptor antagonist. Using a 40 kDa PEGylated (PEG) and half-life extended form of FGF21 (FGF21-PEG) we extended these in vitro results to two different mouse models of diabetes. FGF21-PEG normalized plasma glucose in streptozotocin-treated mice, a model of type 1 diabetes (T1D), without restoring pancreatic β-cell function. FGF21-PEG also normalized plasma glucose levels and improved glucose tolerance in mice chronically treated with an insulin competitive insulin receptor antagonist, a model of autoimmune/Type-B insulin resistance. These data extend the pharmacological potential of FGF21 beyond the settings of T2D, fatty liver and obesity
Exploring the Chemical Space of Protein Glycosylation in Noncovalent Protein Complexes: An Expedition along Different Structural Levels of Human Chorionic Gonadotropin by Employing Mass Spectrometry.
No full textModern analytical approaches employing high-resolution mass spectrometry (MS) facilitate the generation of a vast amount of structural data of highly complex glycoproteins. Nevertheless, systematic interpretation of this data at different structural levels remains an analytical challenge. The glycoprotein utilized as a model system in this study, human chorionic gonadotropin (hCG), exists as a heterodimer composed of two heavily glycosylated subunits. In order to unravel the multitude of glycoforms of recombinant hCG (drug product Ovitrelle), we combine established techniques, such as released glycan and glycopeptide analysis, with novel approaches employing high-performance liquid chromatography-mass spectrometry (HPLC-MS) to characterize protein subunits and native MS to analyze the noncovalent hCG complex. Starting from the deconvoluted mass spectrum of dimeric hCG comprising about 50 signals, it was possible to explore the chemical space of hCG glycoforms and elucidate the complexity that hides behind just 50 signals. Systematic, stepwise integration of data obtained at the levels of released glycans, glycopeptides, and subunits using a computational annotation tool allowed us to reveal 1031 underlying glycoforms. Additionally, critical quality attributes such as sialylation and core fucosylation were compared for two batches of Ovitrelle to assess the potential product variability
A Microphysiological Cell-Culturing System for Pharmacokinetic Drug Exposure and High-Resolution Imaging of Arrays of 3D Microtissues.
No full textUnderstanding the pharmacokinetic/pharmacodynamic (PK/PD)-relationship of a drug candidate is key to determine effective, yet safe treatment regimens for patients. However, current testing strategies are inefficient in characterizing in vivo responses to fluctuating drug concentrations during multi-day treatment cycles. Methods based on animal models are resource-intensive and require time, while traditional in vitro cell-culturing methods usually do not provide temporally-resolved information on the effects of in vivo-like drug exposure scenarios. To address this issue, we developed a microfluidic system to 1) culture arrays of three-dimensional spheroids in vitro, to 2) apply specific dynamic drug exposure profiles, and to 3) in-situ analyze spheroid growth and the invoked drug effects in 3D by means of 2-photon microscopy at tissue and single-cell level. Spheroids of fluorescently-labeled T-47D breast cancer cells were monitored under perfusion-culture conditions at short time intervals over three days and exposed to either three 24 h-PK-cycles or a dose-matched constant concentration of the phosphatidylinositol 3-kinase inhibitor BYL719. While the overall efficacy of the two treatment regimens was similar, spheroids exposed to the PK profile displayed cycle-dependent oscillations between regression and regrowth. Spheroids treated with a constant BYL719 concentration regressed at a steady, albeit slower rate. At a single-cell level, the cell density in BYL719-treated spheroids oscillated in a concentration-dependent manner. Our system represents a versatile tool for in-depth preclinical characterization of PK/PD parameters, as it enables an evaluation of drug efficacy and/or toxicity under realistic exposure conditions
Alternative Complement Pathway Inhibition Abrogates Pneumococcal Opsonophagocytosis in Vaccine-Naïve, but Not in Vaccinated Individuals
No full textTo assess the relative contribution of opsonisation by antibodies, classical and alternative complement pathways to pneumococcal phagocytosis, we analyzed killing of pneumococci by human blood leukocytes collected from vaccine-naïve and PCV13-vaccinated subjects. With serotype 4 pneumococci as model, two different physiologic opsonophagocytosis assays based on either hirudin-anticoagulated whole blood or on washed cells from EDTA-anticoagulated blood reconstituted with active serum, were compared. Pneumococcal killing was measured in the presence of inhibitors targeting the complement components C3, C5, MASP-2, factor B or factor D. The two assay formats yielded highly consistent and comparable results. They highlighted the importance of alternative complement pathway activation for efficient opsonophagocytic killing in blood of vaccine-naïve subjects. In contrast, alternative complement pathway inhibition did not affect pneumococcal killing in PCV13-vaccinated individuals. Independent of amplification by the alternative pathway, even low capsule-specific antibody concentrations were sufficient to efficiently trigger classical pathway mediated opsonophagocytosis. In heat-inactivated or C3-inhibited serum, high concentrations of capsule-specific antibodies were required to trigger complement-independent opsonophagocytosis. Our findings suggest that treatment with alternative complement pathway inhibitors will increase susceptibility for invasive pneumococcal infection in non-immune subjects, but it will not impede pneumococcal clearance in vaccinated individuals
Use of Green Solvents in Metallaphotoredox Cross-Electrophile Coupling Reactions Utilizing a Lipophilic Modified Dual Ir/Ni Catalyst System
No full textFacilitating photoredox coupling reactions in process-friendly green solvents was achieved by the successful application of a dual Ir/Ni catalyst system with enhanced solubility properties. These photochemical reactions (specifically Br–Br sp2–sp3 cross electrophile coupling) are reported in a head to head comparison to the standard di-t-Bu bipyridine ligand Ir/Ni catalyst system. This presentation highlights the benefits of altering the solubility properties of the ligands used in the Ir/Ni dual catalyst
Regioselective Amination or Alkoxylation of Halogenated Amino-, Thio- or Alkoxypyridines via Pyridyne Intermediates
No full textThe treatment of 3-halopyridines (Cl, Br) bearing an R-substituent in position 2 (R = OEt, NEt2, N-piperidyl, or SEt) or in position 5 (R = OMe, OEt, SEt, NMe2, NEt2, or aryl) with KHMDS and an amine at 25 °C for 12 hours in THF provided regioselectively 3- and 4-aminated pyridines in 56–90% yields. The reaction of 3-bromo-2-diethylaminopyridine with various alcohols in the presence of t-BuOK/18-crown-6 in THF at 80 °C for 20–60 hours gave various 4-alkoxy-2-diethylaminopyridines in 61–81% yields. These substitution reactions were proposed to proceed via pyridyne intermediates
Dynamic single-cell RNA sequencing identifies immunotherapy persister cells following PD-1 blockade.
No full textResistance to oncogene-targeted therapies involves discrete drug-tolerant persister cells, originally discovered through in vitro assays. Whether a similar phenomenon limits efficacy of programmed cell death 1 (PD-1) blockade is poorly understood. Here, we performed dynamic single-cell RNA-Seq of murine organotypic tumor spheroids undergoing PD-1 blockade, identifying a discrete subpopulation of immunotherapy persister cells (IPCs) that resisted CD8+ T cell-mediated killing. These cells expressed Snai1 and stem cell antigen 1 (Sca-1) and exhibited hybrid epithelial-mesenchymal features characteristic of a stem cell-like state. IPCs were expanded by IL-6 but were vulnerable to TNF-α-induced cytotoxicity, relying on baculoviral IAP repeat-containing protein 2 (Birc2) and Birc3 as survival factors. Combining PD-1 blockade with Birc2/3 antagonism in mice reduced IPCs and enhanced tumor cell killing in vivo, resulting in durable responsiveness that matched TNF cytotoxicity thresholds in vitro. Together, these data demonstrate the power of high-resolution functional ex vivo profiling to uncover fundamental mechanisms of immune escape from durable anti-PD-1 responses, while identifying IPCs as a cancer cell subpopulation targetable by specific therapeutic combinations
Patient-centric design for systemic peptide delivery: Trends in routes of administration and advancement in drug delivery technologies
No full textUtilizing peptides as therapeutic agents is considered an attractive approach for the treatment of various diseases due to their potential to offer high binding and selectivity to novel drug targets. However, the physicochemical properties of peptides make them challenging to administer exogenously, which places a high burden on drug delivery technology selection to successfully achieve the desired response. Various peptide drug delivery systems have been used in clinical and marketed products to overcome the problems associated with exogenous administration of peptide therapeutics. This review details the trends observed pertaining to the delivery of peptides such as changes in the routes of administration, advancements in formulation platforms, patient-centric product design, and various other aspects of peptide delivery