7196 research outputs found
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Novel biomarkers for glycaemic deterioration in type 2 diabetes: an IMI RHAPSODY study
No full textWe have deployed a multiomics approach in large cohorts of patients with existing type 2 diabetes to identify biomarkers for disease progression across three molecular classes, metabolites, lipids and proteomics. A Cox regression analysis for correlation with time-to-insulin in 5,953 patients in the DCS, ANDIS and GoDARTS cohorts identified homocitrulline, isoleucine and 2-aminoadipic acid, as well as the bile acids glycocholic and taurocholic acids, as predictive of more rapid deterioration. Increased levels of eight triacylglycerol species, and lowered levels of the sphingomyelin SM 42:2:2 were also predictive of disease progression. Of ~1,300 proteins examined in DCS and GoDARTS, levels of GDF-15/MIC1, IL-18RA, CRELD1, NogoR, FAS, and ENPP7 were positively correlated with progression, whilst SMAC/DIABLO, COTL1, SPOCK1 and HEMK2 predicted lower progression rates. Implicating roles in disease compensation, NogoR/RTN4R improved glucose tolerance in high fat-fed mice and tended to improved insulin signalling in liver cells whilst IL18R antagonised inflammatory IL-18 signalling towards nuclear factor kappa-B in vitro. Conversely, high NogoR levels led to islet cell apoptosis, This comprehensive, multi-disciplinary approach thus identifies novel biomarkers with potential diagnostic utility, provides evidence for new disease mechanisms, and identifies potential therapeutic avenues to slow diabetes progression
Real-time imaging of monoclonal antibody film reconstitution after mechanical stress at the air-liquid interface by Brewster angle microscopy.
No full textMonoclonal antibodies (mAbs) represent an important part of biological pharmaceutics. A serious challenge in their development is the formation of protein particles, which are often formed through protein aggregation at the air-liquid interface and then introduced into solution by interfacial stresses. In this paper, protein films formed at the air-liquid interface by two mAbs were disrupted by puncturing them with a microscopic needle, and the subsequent reconstitution of the film was observed in real-time by Brewster angle microscopy. Our results indicate that film reconstitution pace depends on mAb bulk concentration. Numerical modeling gives a quantitative prediction of the surface reconstitution. By extrapolating the model to concentrations typical for pharmaceutical formulations (>30 mg/mL) reconstitution timescales of the protein films can be estimated to be shorter than 0.01 s. Moreover, the effect of polysorbate 80 addition on protein film was studied. Film reconstitution measurements revealed that polysorbate 80 inhibits the film reconstitution process and breaks up the previously formed film
Genotoxicity evaluation of a valsartan-related complex N-nitroso-impurity
No full textRecently, the formation of genotoxic and carcinogenic N-nitrosamines impurities during drug manufacturing has been described. However, drug-related (complex) nitrosamines may also be generated under certain conditions, i.e., through nitrosation of vulnerable amines in drug substances in the presence of nitrite. An investigation of valsartan drug substance showed that a complex API-related N-nitrosamine chemically designated as (S)-2-(((2'-(1H-tetrazol-5-yl)-[1,1'-biphenyl]-4-yl)methyl)(nitroso)amino)-3-methylbutanoic acid (named 181-14) may be generated. 181-14 was shown to be devoid of a mutagenic potential in the Ames test. According to ICH M7 (R1) (2018), impurities that are not mutagenic in the Ames test would be considered Class 5 impurities and limited according to ICH Q3A and B (R2) (2006) guidelines. However, certain regulatory authorities raised the concern that the Ames test may not be sufficiently sensitive to detect a mutagenic potential of nitrosamines and requested a confirmatory in vivo study using a transgenic animal genotoxicitymodel. Our data show that 181-14 was not mutagenic in the transgenic gene mutation assay in MutaTMMice. The data support the conclusion that the Ames test is an adequate and sensitive test system to assess a mutagenic potential of nitrosamines
Principles and Procedures for Assessment of Acute Toxicity Incorporating In Silico Methods
No full textAcute toxicity in silico models are being used to support an increasing number of application areas including (1) product research and development, (2) product approval and registration as well as (3) the transport, storage and handling of chemicals. The adoption of such models is being hindered, in part, because of a lack of guidance describing how to perform and document an in silico analysis. To address this issue, a framework for an acute toxicity hazard assessment is proposed. This framework combines results from different sources including in silico methods and in vitro or in vivo experiments. In silico methods that can assist the prediction of in vivo outcomes (i.e., LD50) are analyzed concluding that predictions obtained using in silico approaches are now well-suited for reliably supporting assessment of LD50-based acute toxicity for the purpose of GHS classification. A general overview is provided of the endpoints from in vitro studies commonly evaluated for predicting acute toxicity (e.g., cytotoxicity/cytolethality as well as assays targeting specific mechanisms). The increased understanding of pathways and key triggering mechanisms underlying toxicity and the increased availability of in vitro data allow for a shift away from assessments solely based on endpoints such as LD50, to mechanism-based endpoints that can be accurately assessed in vitro or by using in silico prediction models. This paper also highlights the importance of an expert review of all available information using weight-of-evidence considerations and illustrates, using a series of diverse practical use cases, how in silico approaches support the assessment of acute toxicity
Smart design of patient centric long-acting products: from preclinical to marketed pipeline trends and opportunities.
No full textWe see a development in the field of long-acting products to serve patients with chronic diseases by providing benefits in adherence, efficacy and safety of the treatment. This review investigates features of long-acting products on the market/pipeline to understand which drug substance (DS) and drug product (DP) characteristics likely enable a successful patient-centric, low-dosing frequency product.This review evaluates marketed/pipeline long-acting products with greater than one week release of small molecules and peptides by oral and injectable route of administration (RoA), with particular focus on patient centricity, adherence impact, health outcomes, market trends, and the match of DS/DP technologies which lead to market success.Emerging trends are expected to change the field of long-acting products in the upcoming years by increasing capability in engineered molecules (low solubility, long half-life, high potency, etc.), directly developing DP as long-acting oral/injectable, increasing the proportion of products for local drug delivery, and a direction towards more subcutaneous, self-administered products. Among long-acting injectable products, nanosuspensions show a superiority in dose per administration and dosing interval, overwhelming the field of infectious diseases with the recently marketed products
Current approaches for predicting human PK for small molecule development candidates: Findings from the IQ Human PK Prediction Working Group Survey
No full textAccurate prediction of human clearance (CL) and volume of distribution at steady state (Vd,ss) for small molecule drug candidates is an essential component of assessing likely efficacious dose and clinical safety margins. In 2021, the IQ Consortium Human PK Prediction Working Group undertook a survey of IQ member companies to understand the current PK prediction methods being used to estimate these parameters across the pharmaceutical industry.
The survey revealed a heterogeneity in approaches being used across the industry (e.g., use of allometric approaches, differing incorporation of binding terms, and inconsistent use of empirical correction factors for in vitro-in vivo extrapolation, IVIVE), which could lead to different PK predictions with the same input data. Member companies expressed an interest in improving human PK predictions by identifying the most appropriate compound-class specific methods, as determined by physiochemical properties and knowledge of CL pathways. Furthermore, there was consensus that increased understanding of the uncertainty inherent to the compound class-dependent prediction would be invaluable in aiding communication of human PK and dose uncertainty at the time of candidate nomination for development.
The human PK Prediction Working Group is utilizing these survey findings to help interrogate clinical IV datasets from across the IQ consortium member companies to understand PK prediction accuracy and uncertainty from preclinical datasets
1H and 19F NMR chemical shifts for hydrogen bond strength determination: Correlations between experimental and computed values
No full text1H and 19F NMR methods based on chemical shift measurements of different hydrogen bond donors used for quantifying hydrogen bond strength were analyzed and compared. The extracted values from these different methods are shown to be highly correlated with each other and with several experimental and ab initio computed quantities characterizing hydrogen bond formation. The titration method based on 19F NMR spectroscopy was performed for detecting and quantifying the formation of very weak hydrogen bond complexes such as those involving fluorine atoms as hydrogen bond acceptors. This approach represents a powerful and reliable method for studying and characterizing these complexes that, although weak, are very relevant
Steric accessibility of the N-terminus improves the titer and quality of recombinant proteins secreted from Komagataella phaffii
No full textBackground: Komagataella phaffii is a commonly used alternative host for manufacturing therapeutic proteins, in part because of its ability to secrete recombinant proteins into the extracellular space. Incorrect processing of secreted proteins by cells can, however, cause non-functional product-related variants, which are expensive to remove in purification and lower overall process yields. The secretion signal peptide, attached to the N-terminus of the recombinant protein, is a major determinant of the quality of the protein sequence and yield. In K. phaffii, the signal peptide from the Saccharomyces cerevisiae alpha mating factor often yields the highest secreted titer of recombinant proteins, but the quality of secreted protein can vary highly.
Results: We determined that an aggregated product-related variant of the SARS-CoV-2 receptor binding domain is caused by N-terminal extension from incomplete cleavage of the signal peptide. We eliminated this variant and improved secreted protein titer up to 76% by extension of the N-terminus with a short, functional peptide moiety or with the EAEA residues from the native signal peptide. We then applied this strategy to three other recombinant subunit vaccine antigens and observed consistent elimination of the same aggregated product-related variant. Finally, we demonstrated that this benefit in quality and secreted titer can be achieved with addition of a single amino acid to the N-terminus of the recombinant protein.
Conclusions: Our observations suggest that steric hindrance of proteases in the Golgi that cleave the signal peptide can cause unwanted N-terminal extension and related product variants. We demonstrated that this phenomenon occurs for multiple recombinant proteins, and can be addressed by minimal modification of the N-terminus to improve steric accessibility. This strategy may enable consistent secretion of a broad range of recombinant proteins with the highly productive alpha mating factor secretion signal peptide.
Keywords: Aggregation; Pichia pastoris; Product quality; Protein engineering; Signal peptide
The Disordered MAX N-terminus Modulates DNA Binding of the Transcription Factor MYC:MAX.
No full textThe intrinsically disordered protein MYC belongs to the family of basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factors (TFs). In complex with its cognate binding partner MAX, MYC preferentially binds to E-Box promotor sequences where it controls fundamental cellular processes such as cell cycle progression, metabolism, and apoptosis. Intramolecular regulation of MYC:MAX has not yet been investigated in detail. In this work, we use Nuclear Magnetic Resonance (NMR) spectroscopy to identify and map interactions between the disordered MAX N-terminus and the MYC:MAX DNA binding domain (DBD). We find that this binding event is mainly driven by electrostatic interactions and that it is competitive with DNA binding. Using NMR spectroscopy and Surface Plasmon Resonance (SPR), we demonstrate that the MAX N-terminus serves to accelerate DNA binding kinetics of MYC:MAX and MAX:MAX dimers, while it simultaneously provides specificity for E-Box DNA. We also establish that these effects are further enhanced by Casein Kinase 2-mediated phosphorylation of two serine residues in the MAX N-terminus. Our work provides new insights how bHLH-LZ TFs are regulated by intramolecular interactions between disordered regions and the folded DNA binding domain
Passivation Effect of Micelles on [Ni-Co] Bimetallic Nanosheets for (Z)-Selective Semihydrogenation of Alkynes Using Water as the Hydrogen Source
No full textA micelle-enabled strategy for the stabilization of early row transition metals nanoparticles in water has been developed. This is achieved by the rational design of pyridine based amphiphilic molecule, PY-750-M. Combination of Ni-Co bimetal-lic nanoparticles and PY-750-M in water resulted in the formation of stable nanosheets in aqueous micellar condition. The catalyst was found to be highly active for water splitting at thermal condition and showcase its application for selective semhydrogenation of alkynes using water as the cleanest hydrogen source. This method was also capable of producing di-deuterated alkenes up to 98% deuterium incorporation. This catalytic system was thoroughly characterized using HRTEM to obtain the particle size, distribution, and morphology. Further, XPS studies were performed to find the oxida-tion states of individual metals. Control experiments revealed the significance of each catalyst component to get desired activity and the crucial role of amphiphile PY-750-M for controlling the reactivity and selectivity. This aqueous strategy is applicable to a broad range of substrates and exhibits excellent functional group tolerance and having 99% selectivity for Z-alkenes. The scalability of this methodology is demonstrated on a gram-scale reaction. This approach represents one of the greenest and sustainable route to access (Z)-alkene or di-deuterated alkene