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The activities of drug inactive ingredients on biological targets
No full textExcipients, considered “inactive ingredients,” are a major component of formulated drugs and play key
roles in their pharmacokinetics. Despite their pervasiveness, whether they are active on any targets
has not been systematically explored. We computed the likelihood that approved excipients would bind
to molecular targets. Testing in vitro revealed 25 excipient activities, ranging from low-nanomolar to
high-micromolar XXXXXX. Another 109 activities were identified by testing against clinical safety targets.
In cellular models, five excipients had fingerprints predictive of system-level toxicity. Exposures of
seven excipients were investigated, and in certain populations, two of these may reach levels of in vitro
target potency, including brain and gut exposure of thimerosal and its major metabolite, which had
dopamine D3 receptor Kd values of 320 and 210 nM, respectively. Although most excipients deserve their
status as inert, many approved excipients may directly modulate physiologically relevant targets
Fokus: Wissenschaftliche Karrierepfade Stefan Ewert: Forschen für die Gesundheit
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Analysis of Live Single Cells by Confocal Microscopy and High-Resolution Mass Spectrometry to Study Drug Uptake, Metabolism, and Drug-Induced Phospholipidosis
No full textThe analysis of large numbers of cells from a population results in information that does not reflect differences in cell phenotypes. Individual variations in cellular drug uptake, metabolism, and response to drug treatment may have profound effects on cellular survival and lead to the development of certain disease states, drug persistence, and resistance. Herein, we present a method that combines live cell confocal microscopy imaging with high-resolution mass spectrometry to achieve absolute cell quantification of the drug amiodarone (AMIO) and its major metabolite, N-desethylamiodarone (NDEA), in single liver cells (HepG2 and HepaRG cells). The method uses a prototype system that integrates a confocal microscope with an XYZ stage robot to image and automatically sample selected cells from a sample compartment, which is kept under growth conditions, with nanospray tips. Besides obtaining the distributions of AMIO and NDEA cell concentrations across a population of individual cells, as well as variabilities in drug metabolism, the effect of these on phospholipidosis and cell morphology was studied. The method was suited to identify subpopulations of cells that metabolized less drug and to correlate cell drug concentrations with cell phospholipid content, cell volume, sphericity, and other cell phenotypic features. Using principal component analysis (PCA), the treated cells could be clearly distinguished from vehicle control cells (0 μM AMIO) and HepaRG cells from HepG2 cells. The potential of using multidimensional and multimodal information collected from single cells to build predictive models for cell classification is demonstrated
Robust step detection from different waist-worn sensor positions – implications for clinical studies
No full textAnalysing human gait with inertial sensors provides valuable insights about a wide range of health impairments, including many musculoskeletal and neurological diseases. A representative and reliable assessment of gait requires continuous monitoring over long periods and ideally takes place in the subjects’ habitual environment (free-living). Inconsistent sensor wearing position can affect gait characterization and influence clinical study results, thus clinical study protocols are typically highly proscriptive, instructing all participants to wear the sensor in a uniform manner. This restrictive approach improves data quality but reduces overall adherence. In this work, we analyse the impact of altering sensor wearing position around the waist on sensor signal and step detection. We demonstrate that an asymmetrically worn sensor leads to additional odd-harmonic frequency components in the frequency spectrum. We propose a robust solution to overcome sensor position variation using autocorrelation. The proposed solution reduces the impact of the sensor positioning on gait characterization in clinical studies, thus providing more flexibility to protocol implementation and more freedom to participants to wear the sensor in the position most comfortable to them. This work is a first step towards truly device-agnostic gait assessment in clinical settings
A cell-level quality control workflow for high-throughput image analysis
No full textBACKGROUND: Image-based high throughput (HT) screening provides a rich source of information on dynamic cellular response to external perturbations. The large quantity of data generated necessitates computer-aided quality control (QC) methodologies to flag imaging and staining artifacts. Existing image- or patch-level QC methods require separate thresholds to be simultaneously tuned for each image quality metric used, and also struggle to distinguish between artifacts and valid cellular phenotypes. As a result, extensive time and effort must be spent on per-assay QC feature thresholding, and valid images and phenotypes may be discarded while image- and cell-level artifacts go undetected. RESULTS: We present a novel cell-level QC workflow built on machine learning approaches for classifying artifacts from HT image data. First, a phenotype sampler based on unlabeled clustering collects a comprehensive subset of cellular phenotypes, requiring only the inspection of a handful of images per phenotype for validity. A set of one-class support vector machines are then trained on each biologically valid image phenotype, and used to classify individual objects in each image as valid cells or artifacts. We apply this workflow to two real-world large-scale HT image datasets and observe that the ratio of artifact to total object area (ARcell) provides a single robust assessment of image quality regardless of the underlying causes of quality issues. Gating on this single intuitive metric, partially contaminated images can be salvaged and highly contaminated images can be excluded before image-level phenotype summary, enabling a more reliable characterization of cellular response dynamics. CONCLUSIONS: Our cell-level QC workflow enables identification of artificial cells created not only by staining or imaging artifacts but also by the limitations of image segmentation algorithms. The single readout ARcell that summaries the ratio of artifacts contained in each image can be used to reliably rank images by quality and more accurately determine QC cutoff thresholds. Machine learning-based cellular phenotype clustering and sampling reduces the amount of manual work required for training example collection. Our QC workflow automatically handles assay-specific phenotypic variations and generalizes to different HT image assays
AXIN2+ Pericentral Hepatocytes Have Limited Contributions to Liver Homeostasis and Regeneration.
No full textThe existence of specialized liver stem cell populations, including AXIN2+ pericentral hepatocytes, that safeguard homeostasis and repair has been controversial. Here, using AXIN2 lineage tracing in BAC-transgenic mice, we confirm the regenerative potential of intestinal stem cells (ISCs) but find limited roles for pericentral hepatocytes in liver parenchyma homeostasis. Liver regrowth following partial hepatectomy is enabled by proliferation of hepatocytes throughout the liver, rather than by a pericentral population. Periportal hepatocyte injury triggers local repair as well as auxiliary proliferation in all liver zones. DTA-mediated ablation of AXIN2+ pericentral hepatocytes transiently disrupts this zone, which is reestablished by conversion of pericentral vein-juxtaposed glutamine synthetase (GS)- hepatocytes into GS+ hepatocytes and by compensatory proliferation of hepatocytes across liver zones. These findings show hepatocytes throughout the liver can upregulate AXIN2 and LGR5 after injury and contribute to liver regeneration on demand, without zonal dominance by a putative pericentral stem cell population
Clinical translation of liver regeneration therapies: A conceptual road map
No full textThe increasing incidence of severe liver diseases worldwide has resulted in a high demand for curative liver transplantation. Unfortunately, the need for transplants by far eclipses the availability of suitable grafts leaving many waitlisted patients to face liver failure and often death. Use of smaller left lobe grafts from living or deceased donors could increase the number of life-saving grafts but is often limited by the graft versus recipient weight ratio defining the safety margins that minimize the risk of small for size syndrome (SFSS. In addition, smaller grafts from deceased donors also face a similar fate. SFSS is a severe complication characterized by failure of a small liver graft to regenerate and occurs when a donor graft is insufficient to meet the metabolic demand of the recipient, leading to liver failure as a result of insufficient liver mass. SFSS is not limited to transplantation but can also occur in the setting of hepatic surgical resections, where life-saving large resections of tumors may be limited by concerns of post-surgical liver failure. There are as yet no available pro-regenerative therapies to enable liver regrowth and thus prevent SFSS. However, there is optimism around targeting factors and pathways that have been identified as regulators of liver regeneration to induce regrowth in-vivo and ex-vivo for clinical use. In this commentary, we propose a roadmap for developing such pro-regenerative therapy and for bringing it into the clinic. We summarize the clinical indications, preclinical models, pro-regenerative pathways and safety considerations necessary for developing such a drug
An RNA polymerase ribozyme that synthesizes its own ancestor
No full textThe RNA-based organisms from which modern life is thought to have descended would have depended on an RNA polymerase ribozyme to copy functional RNA molecules, including copying the polymerase itself. Such a polymerase must have been capable of copying structured RNAs with high efficiency and high fidelity to maintain genetic information across successive generations. Here the class I RNA polymerase ribozyme was evolved in vitro for the ability to synthesize functional ribozymes, resulting in the markedly improved ability to synthesize complex RNAs using nucleoside 5'-triphosphate (NTP) substrates. The polymerase is descended from the class I ligase, which contains the same catalytic core as the polymerase. The class I ligase can be synthesized by the improved polymerase as three separate RNA strands that assemble to form a functional ligase. The polymerase also can synthesize the complement of each of these three strands. Despite this remarkable level of activity, only a very small fraction of the assembled ligases retain catalytic activity due to the presence of disabling mutations. Thus, the fidelity of RNA polymerization should be considered a major impediment to the construction of a self-sustained, RNA-based evolving system. The propagation of heritable information requires both efficient and accurate synthesis of genetic molecules, a requirement relevant to both laboratory systems and the early history of life on Earth
T cell epitope mapping of secukinumab and ixekizumab in healthy donors
No full textSecukinumab, a human monoclonal antibody that selectively neutralizes IL-17A, has consistently shown low
anti-drug antibody responses in patients with psoriasis, psoriatic arthritis, and ankylosing spondylitis.
Secukinumab has also shown lower in vitro immunogenicity potential compared with other monoclonal
antibodies used to treat psoriasis and psoriatic arthritis, and a significantly lower in vitro T cell precursor
frequency compared with ixekizumab, which targets the same antigen. Here, secukinumab and ixekizumab
were further examined regarding their specific T cell epitopes. Secukinumab- or ixekizumab-specific CD4 T
cell lines were generated from 31 healthy, treatment-naïve donors via 28-day co-culture with mature
monocyte-derived dendritic cells exposed to either antibody. Consistent with previous data, the frequency
of preexisting T cells to secukinumab was significantly lower as compared with ixekizumab. Only two T cell
lines from two different donors could be derived for secukinumab, but no specific T cell epitope was
identified. In contrast, 32 T cell lines from eight donors were obtained for ixekizumab. For 11 of these T cell lines, the specific T cell epitopes could be identified and confirmed by major histocompatibility complex–associated peptide proteomics as being naturally presented peptides. All identified T cell Epitopes cluster in four main regions that are overlapping with the complementarity-determining regions HCDR3, LCDR1, LCDR2 and LCDR3. Interestingly, ixekizumab CDRs contain amino acids that are not found in any of the germline family members. These amino acids may be associated with the higher number of T cell epitopes identified for ixekizumab light chain and may contribute to the increased in vitro immunogenicity potential observed for ixekizumab vs. secukinumab
Discovery of LOU064 (Remibrutinib), a Potent and Highly Selective Covalent Inhibitor of Bruton’s Tyrosine Kinase
No full textBruton’s tyrosine kinase (BTK), a cytoplasmic tyrosine kinase, plays a central role in immunity and is considered an attractive target for treating autoimmune diseases. The use of currently marketed covalent BTK inhibitors is limited to oncology indications based on their suboptimal kinase selectivity. We describe the discovery and preclinical profile of LOU064 (25), a potent, highly selective covalent BTK inhibitor. LOU064 exhibits an exquisite kinase selectivity due to binding to an inactive conformation of BTK and has the potential for a best-in-class covalent BTK inhibitor for the treatment of autoimmune diseases. It demonstrates potent in vivo target occupancy with an EC90 of 1.6 mg/kg and dose-dependent efficacy in rat collagen-induced arthritis. LOU064 is currently being tested in Phase 2 clinical studies for chronic spontaneous urticaria and Sjoegren’s Syndrome