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Constitutive silencing of LRRK2 kinase activity leads to early glucocerebrosidase deregulation and late impairment of autophagy in vivo
No full textMutations in leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease. LRRK2 modulates the autophagy-lysosome pathway (ALP), a clearance process subserving the quality control of cellular proteins and organelles. Since dysfunctional ALP might lead to α-synuclein accumulation and, hence, Parkinson's disease, LRRK2 kinase modulation of ALP, its age-dependence and relation with pSer129 α-synuclein inclusions were investigated in vivo. Striatal ALP markers were analyzed by Western blotting in 3, 12 and 20-month-old LRRK2 G2019S knock-in mice (bearing enhanced kinase activity), LRRK2 knock-out mice, LRRK2 D1994S knock-in (kinase-dead) mice and wild-type controls. The lysosomotropic agent chloroquine was used to investigate the autophagic flux in vivo. Quantitative Real-time PCR was used to quantify the transcript levels of key ALP genes. The activity of the lysosomal enzyme glucocerebrosidase was measured using enzymatic assay. Immunohistochemistry was used to co-localize LC3B puncta with pSer129 α-synuclein inclusion in striatal and nigral neurons. No genotype differences in ALP markers were observed at 3 months. Conversely, increase of LC3-I, p62, LAMP2 and GAPDH levels, decrease of p-mTOR levels and downregulation of mTOR and TFEB expression was observed in 12-month-old kinase-dead mice. The LC3-II/I ratio was reduced following administration of chloroquine, suggesting a defective autophagic flux. G2019S knock-in mice showed LAMP2 accumulation and downregulation of ALP key genes MAP1LC3B, LAMP2, mTOR, TFEB and GBA1. Subacute administration of the LRRK2 kinase inhibitor MLi-2 in wild-type and G2019S knock-in mice did not replicate the pattern of kinase-dead mice. Lysosomal glucocerebrosidase activity was increased in 3 and 12-month-old knock-out and kinase-dead mice. LC3B puncta accumulation and pSer129 α-synuclein inclusions were dissociated in striatal neurons of kinase-dead and G2019S knock-in mice. We conclude that constitutive LRRK2 kinase silencing results in early deregulation of GCase activity followed by late impairment of macroautophagy and chaperone-mediated autophagy
Meeting report of the first European Biotransformation Workshop
No full textChallenges and opportunities in the field of biotransformation were presented and discussed at the 1st European Biotransformation workshop which was conducted virtually in collaboration with the DMDG January 27, 2021. Here we summarise the presentations and discussions from this workshop.
The following topics were covered:
• Needs for radiolabel for IND filing versus quantitation without standards
• Applications of cyclic ion mobility in the field of biotransformation
• Computational predictions of xenobiotic metabolism
• Future (outsourcing) needs in biotransformation
• Genotoxicity risk assessment of metabolites and qualification of impurities using metabolite data
• Regulatory aspects of MIST
Keywords: Biotransformation, metabolite qualification, cyclic ion mobility, MIST, genotoxicity risk assessment, qualification of impuritie
Scales, belts, and pyramids: Back to the future of digital healthcare
No full textAdoption of digital health technologies has been relatively limited despite their potential to have meaningful impact on health. For deployment to improve patient adherence, it is imperative to link desired behavioral changes to patient value chains, many of which are instilled at an early age. There exists a clear opportunity for parents, schools, and communities to play a mentoring role in the deployment of digital health tools by future generations before they become patients. In addition, for existing adult populations and seniors, appropriate motivating factors can be deployed to inspire effective use of digital adherence tools and contribute to enhanced health outcomes in the immediate future. Coupled with new approaches in managed care the full promise of digital health technologies might then be realized in the nearer term
Inspiration and Exasperation: The Challenges of Inhaled Biologics
No full textThe delivery of biotherapeutic molecules (antibodies, proteins, peptides) and nucleic acids via the respiratory route has presented challenges for regulatory approval, due in part to a lack of understanding of the expected pathology, mechanisms of toxicity, and immunogenicity induced by the inhalation route. Although the first inhaled biotherapeutic was approved some time ago (Dornase Alfa, Pulmozyme; Genetech, 1993), no other inhaled biotherapeutics have been marketed for the treatment of human disease other than the inhaled insulins (Exubera; Pfizer, 2006 and Afrezza; Mannkind Corporation, 2014). As a result, scientific knowledge within the toxicologic pathology community is fragmented with precious little publicly available data. Therefore, one of the aims of this special edition was to generate a collection of manuscripts that pathologists and toxicologists could refer in order to understand the pathology, mechanisms of toxicity, immunogenicity, and challenges associated with the development of inhaled biotherapeutics
BSTP Review of 12 Case Studies Discussing the Challenges, Pathology, Immunogenicity, and Mechanisms of Inhaled Biologics
No full textThe inhalation route is a relatively novel drug delivery route for biotherapeutics and, as a result, there is a paucity of published data and experience within the toxicology/pathology community. In recent years, findings arising in toxicology studies with inhaled biologics have provoked concern and regulatory challenges due, in part, to the lack of understanding of the expected pathology, mechanisms, and adversity induced by this mode of delivery. In this manuscript, the authors describe 12 case studies, comprising 18 toxicology studies, using a range of inhaled biotherapeutics (monoclonal antibodies, fragment antigen-binding antibodies, domain antibodies, therapeutic proteins/peptides, and an oligonucleotide) in rodents, nonhuman primates (NHPs), and the rabbit in subacute (1 week) to chronic (26 weeks) toxicology studies. Analysis of the data revealed that many of these molecules were associated with a characteristic pattern of toxicity with high levels of immunogenicity. Microscopic changes in the airways consisted of a predominantly lymphoid perivascular/peribronchiolar (PV/PB) mononuclear inflammatory cell (MIC) infiltrate, whereas changes in the terminal airways/alveoli were characterized by simple (“uncomplicated”) increases in macrophages or inflammatory cell infiltrates ranging from mixed inflammatory cell infiltration to inflammation. The PV/PB MIC changes were considered most likely secondary to immunogenicity, whereas simple increases in alveolar macrophages were most likely secondary to clearance mechanisms. Alveolar inflammatory cell infiltrates and inflammation were likely induced by immune modulation or stimulation through pharmacologic effects on target biology or type III hypersensitivity (immune complex disease). Finally, a group of experts provide introductory thoughts regarding the adversity of inhaled biotherapeutics and the basis for reasonable differences of opinion that might arise between toxicologists, pathologists, and regulators
Digital biomarkers enable automated, longitudinal monitoring in a mouse model of aging
No full textTo understand the growing needs of an aging human population, there is demand for scalable and reproducible approaches to study animal models of aging and to test novel therapeutic interventions. We investigated the sensitivity and utility of a continuous monitoring platform and its digital biomarkers (motion, breathing rate, and wheel running) to evaluate behavioral and physiological differences between "young" (12 weeks) and "old" (23 months) male C57BL/6J mice with or without running wheels in the home cage. Compared to young mice, old mice showed marked reductions in motion and breathing rate, as well as altered circadian rhythms. Mice without running wheels possessed lower breathing rates compared to their counterparts with running wheels. Digital biomarkers showed age-dependent changes in response to routine procedures (cage changes and blood sampling) and alterations in subjects that unexpectedly reached endpoint. Continuous collection of digital biomarkers in the home cage can enhance current approaches by providing unbiased longitudinal monitoring for large-scale aging studies
The biogenesis and function of nucleosome arrays
No full textNumerous chromatin remodeling enzymes position nucleosomes in eukaryotic cells. Aside from these factors, transcription, DNA sequence, and statistical positioning of nucleosomes also shapes the nucleosome landscape. Precise contributions of these processes remain unclear due to their functional redundancy in vivo. By incisive genome engineering, we radically decreased their redundancy in Saccharomyces cerevisiae. The transcriptional machinery is strongly disruptive of evenly spaced nucleosomes, and proper nucleosome density and DNA sequence critical for their biogenesis. The INO80 remodeling complex spaces nucleosomes in vivo and positions the first nucleosome over genes in an H2A.Z-independent fashion. INO80 requires its Arp8 subunit but unexpectedly not the Nhp10 module for spacing. Spaced nucleosomes prevent cryptic transcription and protect cells against genotoxic stress such as DNA damage, recombination and transpositions. We derive a unifying model of the biogenesis of the nucleosome landscape and suggest that it evolved not only to regulate but also to protect the genome
Nanochannels in Photoactive Polymeric Cu(I) Compatible for Efficient Micellar Catalysis: Sustainable Aerobic Oxidations of Alcohols in Water
No full textUpon visible-light irradiation, the heterogeneous polymer of PDI-Cu(I)-PDI generates charge transfer states that are
subsequently quenched by molecular oxygen for their participation in redox activity. This insoluble polymeric Cu(I) is catalytically active for the oxidation of alcohols to aldehyde when suspended in dynamic micelles of PS-750-M. A broad substrate scope, excellent selectivity, and no over-oxidation reveal the catalyst robustness. The catalytic activity, control experiments, and time-dependent DFT calculations show the charge transfer states. The polymeric catalyst is entirely recyclable, as evidenced by the recycle studies using Scott's recyclability test. The morphology, structure, copper's oxidations state, and the catalyst's thermal stability are determined by SEM, XPS, and TGA analysis
Regional brain mGluR5 receptor occupancy following single oral doses of mavoglurant as measured by [11C]-ABP688 PET imaging in healthy volunteers
No full textMavoglurant binds to same allosteric site on metabotropic glutamate receptor 5 (mGluR5) as [11C]-ABP688, a radioligand. This open-label, single-center pilot study estimates extent of occupancy of mGluR5 receptors following single oral doses of mavoglurant, using [11C]-ABP688 positron emission tomography (PET) imaging, in six healthy males aged 20–40 years. This study comprised three periods and six subjects were divided into two cohorts. On Day 1 (Period 1), baseline clinical data and safety samples were obtained along with PET scan. During Period 2 (1–7 days after Period 1), cohort 1 and 2 received mavoglurant 25 mg and 100 mg, respectively. During Period 3 (7 days after Period 2), cohort 1 and 2 received mavoglurant 200 mg and 400 mg, respectively. Mavoglurant showed the highest distribution volumes in the cingulate region with lower uptake in cerebellum and white matter, possibly because myelinated axonal sheets maybe devoid of mGluR5 receptors. Maximum concentrations of mavoglurant were observed around 2–3.25 h post-dose. Mavoglurant passed the blood–brain barrier and induced dose- and exposure-dependent displacement of [11C]-ABP688 from the mGluR5 receptors, 3–4 h post-administration (27%, 59%, 74%, 85% receptor occupancy for mavoglurant 25 mg, 100 mg, 200 mg, 400 mg dose, respectively). There were no severe adverse effects or clinically significant changes in safety parameters. This study can assist in defining a relevant clinical dose range for use in clinical trials, and shows that PET and [11C]-ABP688 provide a unique tool to study drug-induced occupancy of mGlu5 receptors in the living human brain
Water waste from surfactant chemistry: risk or opportunity?
No full textMost biotransformations have been mediated in water as the bulk medium, usually with the addition of surfactants as solubility enhancers. Scattered reports and research interest of selected groups have also driven the usage of water in chemical transformations. But it is not until the advent of Phase-Transfer Catalysis that water could be envisioned as a medium of choice, if not entirely, at least partially. Closer to us, Lipshutz’ seminal report on the use of benign by design surfactant to conduct a large variety of transformations sparked our attention and led us as early as 2011 to aggressively enter the field. We rapidly saw in it significant opportunities from a sustainability standpoint especially, with the prospect of establishing a new paradigm where most chemical transformations could be run sustainably and advantageously in water. With the support of our academic partners, Professor Lipshutz and Sachin Handa, a large and continuously growing number of synthetic methodologies were established to conduct the most important transformations of our portfolio in water, and have been implemented on scale in our plants. An immediate aspect that we needed to address became that of the waste water management. Indeed, while we had demonstrated the absolute needs in water to be lesser than compared to traditional process in organic solvents, we still needed to demonstrate that the water could be disposed safely and that no inherent risk could occur due to the usage of the surfactant. The following manuscript describes our approach to the problem and our recommendations to control the quality of our waste water, and dispose of it in the most sustainable manner