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Flow cytometric method transfer: Recommendations for best practice
No full textAs with many aspects of the validation and monitoring of flow cytometric methods, the method transfer processes and acceptance criteria described for other technologies are not fully applicable. This is due to the complexity of the highly configurable instrumentation, the complexity of cellular measurands, the lack of qualified reference materials for most assays, and limited specimen stability. There are multiple reasons for initiating a method transfer, multiple regulatory settings, and multiple context of use. All of these factors influence the specific requirements for the method transfer. This recommendation paper describes the considerations and best practices for the transfer of flow cytometric methods and provides individual case studies as examples. In addition, the manuscript emphasizes the importance of appropriately conducting a method transfer on data reliability
Continuous Flow Suzuki-Miyaura Couplings Under Aqueous Micellar Conditions in a CSTR Cascade Catalyzed by Fe/ppm Pd Nanoparticles
No full textChemistry in water, enabled by tailor-made surfactants that form nanomicelles of a particular size and shape, has been developed to accommodate flow conditions using Fe/ppm Pd nanoparticles as catalyst for Suzuki-Miyaura reactions. Cross-couplings of this type that are heterogeneous in terms of both the substrate and catalyst are particularly troublesome under flow conditions, and pose a major challenge in continuous manufacturing of pharmaceuticals and fine chemicals. To address this major issue, a new continuous stirred-tank reactor (CSTR) cascade has been engineered that can handle slurries/solids at any point during a chemical transformation in flow. Solid starting materials are now simply suspended in water and pumped into the CSTR as a heterogeneous slurry, minimizing the upstream need for organic solvents, thereby leading to a far more environmentally friendly process. The ability of CSTR cascade to handle solids continuously was demonstrated over hours, without clogging, by examining several couplings which involve both initial solid catalyst and substrates, while generating solids products, some of which are key intermediates in pharmaceuticals currently in use
Topoisomerase Inhibitors Addressing Fluoroquinolone Resistance in Gram-Negative Bacteria
No full textSince their discovery over five decades ago, quinolone antibiotics have found enormous success as broad spectrum agents that exert their activity through dual inhibition of bacterial DNA gyrase and topoisomerase IV. Increasing rates of resistance, driven largely by target-based mutations in the GyrA/ParC Quinolone Resistance Determining Region, have eroded the utility and threaten the future use of this vital class of antibiotics. Herein we describe the discovery and optimization of a series of 4-(aminomethyl)quinolin-2(1H)-ones, exemplified by 34, that inhibit bacterial DNA gyrase and topoisomerase IV and display potent activity against ciprofloxacin-resistant Gram-negative pathogens. X-ray crystallography reveals that 34 occupies the classical quinolone binding site in the topoisomerase IV-DNA cleavage complex, but does not form significant contacts with residues in the Quinolone Resistance Determining Region
Spartalizumab in Combination With Dabrafenib And Trametinib in Advanced BRAF V600–Mutant Melanoma: Efficacy, Safety, and Biomarker Findings in Parts 1 and 2 of COMBI-i
No full textWhile recent 5-year analyses of Phase III trials in metastatic melanoma have demonstrated that checkpoint inhibitors and targeted therapy lead to long-term survival, many patients experience progression and novel treatment strategies are needed. In parts 1 and 2 of COMBI-i (NCT02967692), spartalizumab + dabrafenib + trametinib demonstrated an objective response rate of 78% (95% CI, 61%-90%) and a complete response rate of 44%. Additionally, exploratory biomarker identified baseline biomarkers as well as on-treatment modulations that may predict patients likely to achieve long-term benefit
Fast energy minimization of the CCDC drug subset structures by molecule-in-cluster computations allows independent structure validation and model completion
No full textOptimizing structures with computations on clusters of molecules permits generation of structure-specific restraints for refinement. Equally importantly, retrospective structure validation and addition of hydrogen atoms consistent with quantum chemistry is possible for experimental structures or the solvent molecules in them, should they be missing in earlier CIF depositions. Revisiting the drug subset structures of the CCDC demonstrates that structure validation through ab initio cluster computations is a tremendous validation tool. The time required for optimization can be similar to the time required to carry out least squares refinement for small-molecule structures, and becomes feasible for large structures. Several questions arise: is it valid to augment experimental structures with structure-specific restraints, ideally through accompanying refinement with computation? Do energy minimized structures (using the experimental determinations as a starting point) still constitute an experimental result? When re-refinement is impossible in retrospect, like for most of the drug-subset molecules, then additional value lies in completion and validation of existing structures so that they are chemically and crystallographically correct, and contain missing water or solvent hydrogen atoms. Our results suggest that retroactive validation and addition of hydrogen atoms becomes possible for the entire Cambridge Structural Database. Generation of database entries of optimized alongside existing structures will provide the flexibility needed to make full
use of the information gained by computation
Degradation of polysorbates 20 and 80 catalysed by histidine chloride buffer
No full textPolysorbates are amphiphilic, non-ionic surfactants, and they represent one of the key components of biopharmaceuticals. They serve as stabilisers, and their degradation can cause particle formation, which has been an industry-wide issue over the past decade. To determine the influence of the buffers most frequently used in biopharmaceuticals on polysorbate degradation, an accelerated stability study was carried out using placebo formulations containing 0.02% polysorbates and 20 mM buffers (pH 5.5, 6.5). These included histidine chloride, sodium citrate, sodium succinate and sodium phosphate buffers. The rate of polysorbate degradation was highest in histidine chloride buffer, and therefore we further focused on the mechanism here. The predominant degradation pathway of polysorbates in this buffer was ester hydrolysis, catalysed by the imidazole moiety of the histidine. Interestingly, the presence of therapeutic proteins in the formulations slowed histidine-catalysed degradation of polysorbates in 50% of cases, with negligible degradation seen otherwise. This emphasises the complex nature of the interactions between the components of biopharmaceutical drug products. Nonetheless, there are disadvantages of using histidine chloride buffers in biopharmaceuticals that contain polysorbates. Careful consideration should be given to selection of excipients used in parenteral formulations, whereby compatibility between buffer and surfactant is of key importance
It’s in Our Blood: A Glimpse of Personalized Medicine
No full textRecent advances in protein profiling technology has facilitated simultaneous measurement of thousands of proteins in large population studies, exposing the depth and complexity of the plasma and serum proteomes. This revealed that proteins in circulation were organized into regulatory modules under genetic control and closely associated with current and future common diseases. Unlike networks in solid tissues, serum protein networks comprise members synthesized across different tissues of the body. Genetic analysis reveals that this cross-tissue regulation of the serum proteome participates in systemic homeostasis and mirrors the global disease state of individuals. Here, we discuss how application of this information in routine clinical evaluations may transform the future practice of medicine
Summer 2020 STP President's Report
No full textSummary activities of the Society of Toxicologic Pathology Executive Committee actions in the past quarter, to be published in the electronic only STP society Newsletter "Scope
Targeting BACE to remodel tumor microenvironment and enhance chemo-sensitivity in metastatic melanoma
No full textBy exploring human biopsies from melanoma patients, we found the presence of protein aggregates specifically spread in metastasis compared to primitive lesions. Secretome analysis of metastatic melanoma cell lines revealed the release of amyloidogenic proteins as PMEL, APLP2, APLP1 and APP into the extracellular space. Proteins involved in amyloid maturation as APOE, QPCT, SORT1, VLDLR and DCT were also detected, attesting the presence of an active amyloidogenic machinery. Herein, we demonstrated that BACE activity regulates aggregates production, and its inhibition affects the shedding of the amyloid related proteins. We showed that BACE sheddome regulates extracellular matrix and activates mechanosignaling i.e. by impacting on YAP nuclear localization. Moreover, BACE inhibition impairs cells proliferation and enhances sensitivity to doxorubicin. These results suggest the importance of amyloids production for tumor survival, highlighting the potential of BACE inhibitor as adjuvant melanoma treatments in the development of novel therapeutic approaches
Development of autotaxin inhibitors: A series of tetrazole cinnamides
No full textA series of inhibitors of Autotaxin (ATX) have been developed from a high throughput screening hit, 1a, which shows an alternative binding mode to known catalytic site inhibitors. Selectivity over the hERG channel and microsomal clearance were dependent on the lipophilicity of the compounds, and this was optimised by reduction of clogD whilst maintaining high affinity ATX inhibition. Compound 15a shows good oral exposure, and concentration dependent inhibition of formation of LPA in vivo, as shown in pharmacokinetic-pharmacodynamic (PK/PD) experiment