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The Young Scientists Network – How the European Federation for Medicinal Chemistry (EFMC) became young again.
No full textThe European Federation for Medicinal Chemistry (EFMC) created the Young Scientists Network (YSN) to support early career medicinal chemists and chemical biologists. By doing this, it addressed the rapid changes taking place in the scientific community and in our society. In particular, the rise of social media, the change in gender balance in the scientists’ population, and the evolution of educational needs. Creating the YSN was also a way to ensure that the next generation of scientists would contribute to shaping EFMC’s strategy, while recognizing and addressing their needs. The YSN was set up as a very dynamic concept, and has now developed to the point where its impact is evident. The activities it promotes complement EFMC’s community support and scientific opportunities, rejuvenating the Federation and preparing it for the future. It also provides opportunities for many brilliant, young scientists, who do not hesitate to invest time and energy in supporting our community and shaping their own future
Assessment of the nucleotide modifications in the high-resolution cryo-electron microscopy structure of the Escherichia coli 50S subunit.
No full textPost-transcriptional ribosomal RNA (rRNA) modifications are present in all organisms, but their exact functional roles and positions are yet to be fully characterized. Modified nucleotides have been implicated in the stabilization of RNA structure and regulation of ribosome biogenesis and protein synthesis. In some instances, rRNA modifications can confer antibiotic resistance. High-resolution ribosome structures are thus necessary for precise determination of modified nucleotides' positions, a task that has previously been accomplished by X-ray crystallography. Here, we present a cryo-electron microscopy (cryo-EM) structure of the Escherichia coli 50S subunit at an average resolution of 2.2 Å as an additional approach for mapping modification sites. Our structure confirms known modifications present in 23S rRNA and additionally allows for localization of Mg2+ ions and their coordinated water molecules. Using our cryo-EM structure as a testbed, we developed a program for assessment of cryo-EM map quality. This program can be easily used on any RNA-containing cryo-EM structure, and an associated Coot plugin allows for visualization of validated modifications, making it highly accessible
7th International Workshop on Genotoxicity Testing: Report of the in vivo strategies working group.
No full textThe working group reached complete or majority agreement on the following:
• Results from TGR and in vivo comet assays for 91 chemicals showed that they have similar ability to detect in vivo genotoxicity per se with bacterial mutagens and Ames-positive carcinogens.
• TGR and comet assay results were not significantly different when compared with IARC Group 1, 2A, and unclassified carcinogens. There were significantly more comet assay positive responses for Group 2B chemicals, and for IARC and unclassified carcinogens combined, however we may expect this since mutation is a sub-set of genotoxicity.
• A liver comet assay combined with the MNviv test would detect in vivo genotoxins that do not exhibit tissue-specific or site-of-contact effects, and is appropriate for routine in vivo testing for genotoxicity.
• Generally for orally administered substances, comet analysis of only one site-of-contact GI tract tissue (stomach or duodenum/jejunum) is required.
• In MNviv tests, evidence of target tissue exposure can be obtained in a number of different ways, as recommended by ICH S2(R1) and EFSA (Hardy et al., 2017).
• Except for special cases the i.p. route is inappropriate for in vivo testing, and for risk evaluations more weight should be given to data from a physiologically relevant administration route.
• The liver MN (LMN) test is sufficiently validated for the development of an OECD guideline. However, the impact of dosing animals >6 weeks of age needs to be evaluated. The GI tract MN test shows promise but needs more validation for an OECD guideline.
• The Pig-a assay detects systemically available mutagens and is a valuable follow-up to in vitro positive results. A new freeze-thaw protocol provides more flexibility. Mutant reticulocyte and erythrocyte frequencies should both be determined. Preliminary data are available for the Pig-a assay in male rat germ cells which require validation including germ cell DNA mutation origin
Adjuvant dabrafenib plus trametinib versus placebo in patients with resected, BRAFV600-mutant, stage III melanoma (COMBI-AD): exploratory biomarker analyses from a randomised, phase 3 trial
No full textBackground
Adjuvant dabrafenib plus trametinib reduced the risk of relapse versus placebo in patients with resected, BRAFV600-mutant, stage III melanoma in the phase 3 COMBI-AD trial. This prespecified exploratory biomarker analysis aimed to evaluate potential prognostic or predictive factors and mechanisms of resistance to adjuvant targeted therapy.
Methods
COMBI-AD is a randomised, double-blind, placebo-controlled, phase 3 trial comparing dabrafenib 150 mg orally twice daily plus trametinib 2 mg orally once daily versus two matched placebos. Study participants were at least 18 years of age and underwent complete resection of stage IIIA (lymph node metastases >1 mm), IIIB, or IIIC cutaneous melanoma, per American Joint Committee on Cancer 7th edition criteria, with a BRAFV600E or BRAFV600K mutation. Patients were randomly assigned (1:1) to the two treatment groups by an interactive voice response system, stratified by mutation type and disease stage. Patients, physicians, and the investigators who analysed data were masked to treatment allocation. The primary outcome was relapse-free survival, defined as the time from randomisation to disease recurrence or death from any cause. Biomarker assessment was a prespecified exploratory outcome of the trial. We assessed intrinsic tumour genomic features by use of next-generation DNA sequencing and characteristics of the tumour microenvironment by use of a NanoString RNA assay, which might provide prognostic and predictive information. This trial is registered with ClinicalTrials.gov, number NCT01682083, and is ongoing but no longer recruiting participants.
Findings
Between Jan 31, 2013, and Dec 11, 2014, 870 patients were enrolled in the trial. Median follow-up at data cutoff (April 30, 2018) was 44 months (IQR 38–49) in the dabrafenib plus trametinib group and 42 months (21–49) in the placebo group. Intrinsic tumour genomic features were assessed in 368 patients (DNA sequencing set) and tumour microenvironment characteristics were assessed in 507 patients (NanoString biomarker set). MAPK pathway genomic alterations at baseline did not affect treatment benefit or clinical outcome. An IFNγ gene expression signature higher than the median was prognostic for prolonged relapse-free survival in both treatment groups. Tumour mutational burden was independently prognostic for relapse-free survival in the placebo group (high TMB, top third; hazard ratio [HR] 0·56, 95% CI 0·37–0·85, p=0·0056), but not in the dabrafenib plus trametinib group (0·83, 95% CI 0·53–1·32, p=0·44). Patients with tumour mutational burden in the lower two terciles seem to derive a substantial long-term relapse-free survival benefit from targeted therapy (HR [versus placebo] 0·49, 95% CI 0·35–0·68, p<0·0001). However, patients with high tumour mutational burden seem to have a less pronounced benefit with targeted therapy (HR [versus placebo] 0·75, 95% CI 0·44–1·26, p=0·27), especially if they had an IFNγ signature lower than the median (HR 0·88 [95% CI 0·40–1·93], p=0·74).
Interpretation
Tumour mutational burden alone or in combination with IFNγ gene expression signature or other markers for an adaptive immune response might be of relevance for identifying patients with stage III melanoma who might derive clinical benefit from targeted therapy. Further validation in prospective clinical trials is warranted
Simplification of FDM 3D-Printing paradigm: feasibility of 1-step Direct Powder Printing for Immediate Release dosage forms production
No full textThree-dimensional (3D)-printing of tablets via fused deposition modelling (FDM) is gaining attention for the production of flexible and personalized dosage forms. FDM presents advantages for decentralized on-site manufacturing in hospitals and pharmacies as no powder or solvents are involved in the printing process and post-processing can be avoided. However, the current FDM paradigm for dosage forms development is complex, and involves a hot-melt extrusion step and 3D printable drug-loaded filaments as intermediate products for tablet manufacturing.
In this study, simplification of the current FDM set-up for rapid release dosage forms manufacturing was explored. Several powder blends were directly loaded into a cartridge-like head and were successfully printed directly with honeycomb design following heating of the extrusion cartridge. This served as a proof of concept for 1-step direct powder printing (DPP) with incorporation of in-built porosity allowing higher surface area.
A heat processable, water soluble polymer, Hydroxypropylcellulose (HPC) SSL was chosen as rapid release matrix former and caffeine (10%) as thermally stable model drug. The effect of incorporation of a plasticizer/pore former (PEG4000) and a rapidly dissolving polymer (Kollidon VA64) on DPP processability and dissolution profiles was investigated. Formulations were directly 3D-printed into solid dosage forms with high (80%) and low (30%) infill density, and critical quality attributes analyzed (e.g. dissolution profiles, chemical stability and physical form).
The obtained directly 3D-printed tablets demonstrated good weight and content uniformity. Low infill density tablets showed rapid release dissolution profiles independently of the formulation, whereas for high infill density tablets a combination of pore-former PEG4000 and rapidly-dissolving polymer Kollidon VA64 was required to achieve rapid release. Caffeine was found in crystalline state and in the desired polymorph in directly 3D-printed tablets.
Direct Powder 3D-printing feasibility for immediate release dosage forms manufacturing was demonstrated. This technique might create an opportunity to skip the hot-melt extrusion step, allowing 3D-printing independent of mechanical properties of a filament. This might potentially prolong formulation shelf life as thermal stress is applied only once, shortly before the tablets production and dispensing. Moreover, this powder-in-a-cartridge technique might create a future opportunity for decentralized production: loading powder formulation in a cartridge at the industrial facility, and 3D-printing on clinical site potentially using in the same 3D-printer for implants, tablets and even tissues and organs
Biology-Inspired Microphysiological Systems to Advance Medicines for Patients Benefit
No full textno abstrac
Efficacy of piperacillin in combination with novel β-lactamase inhibitor IID572 against β-lactamase-producing strains of Enterobacteriaceae and Staphylococcus aureus in murine neutropenic thigh infection models
No full textObjectives: The neutropenic murine thigh infection model was used to assess the effectiveness of IID572, a novel β-lactamase inhibitor, in rescuing piperacillin activity against bacterial strains expressing various β-lactamase enzymes.
Methods: Mice (n = 4/group) were inoculated with Enterobacteriaceae or Staphylococcus aureus bacterial strains expressing a range of β-lactamases via intramuscular injection. Two hours after bacterial inoculation, subcutaneous treatment with piperacillin/IID572 or piperacillin/tazobactam every 3 h was initiated. Animals were euthanized via CO2 24 h after the start of therapy and bacterial cfu (log10 cfu) per thigh was determined, and the static dose was calculated.
Results: In a dose-dependent manner, piperacillin/IID572 reduced the thigh bacterial burden in models established with Enterobacteriaceae producing class A, C and D β-lactamases (e.g. ESBLs, KPC, CMY-2 and OXA-48). Piperacillin/IID572 was also efficacious against MSSA strains, including one producing β-lactamase. Static doses of piperacillin/IID572 were calculable from animals infected with all strains tested and the calculated static doses ranged from 195 to 4612 mg/kg/day piperacillin, the active component in the combination. Of the 13 strains investigated, a 1 log10 bacterial reduction was achieved for 9 isolates and a 2 log10 reduction was achieved for 3 isolates; piperacillin/tazobactam was not efficacious against 6 of the 13 isolates tested.
Conclusions: In contrast to tazobactam, IID572 was able to rescue piperacillin efficacy in murine thigh infection models established with β-lactamase-producing strains of Enterobacteriaceae and S. aureus, including those expressing ESBLs or serine carbapenemases
A high throughput screening identifies MICU1 targeting compounds
No full textMitochondrial Ca2+ uptake depends on the mitochondrial calcium uniporter (MCU), a highly selective tetrameric channel of the inner mitochondrial membrane (IMM), composed of pore forming and of regulatory subunits. Here, we screened a library of 44k non-proprietary compounds for their ability to modulate mitochondrial Ca2+ uptake. Two of them decreased mitochondrial Ca2+ influx both in cell lines and in isolated mouse skeletal muscle fibers. A closer inspection revealed that these molecules directly bound a specific cleft in the MCU complex component and positive regulator MICU1. In MICU1-silenced or deleted cells, the inhibitory effect of the two compounds on Ca2+ influx was lost, demonstrating that MICU1 is required for compound activity. Moreover, in MICU1-KO cells overexpressing a mutant isoform of MICU1, in which critical amino acids of the predicted binding cleft were mutated, the two compounds were unable to decrease mitochondrial Ca2+ uptake. Finally, the compounds were tested ex vivo unravelling a role for mitochondrial Ca2+ uptake in muscle growth. These compounds represent leading molecules for the development of MICU1-targeting drugs
Structural Analysis of Metastable Pharmaceutical Loratadine Form II, by 3D Electron Diffraction and DFT+D Energy Minimisation
No full textMetastable polymorphs typically display higher solubility than their thermodynamically stable counterparts, whilst having dissimilar mechanical and biopharmaceutical properties. It is unsurprising then, that generic and innovator companies alike pursue isolation and characterisation of these materials. Here we report the determination of the crystal structure of the metastable Form II of loratadine using a combination of low-resolution 3D electron diffraction data and density functional theory. Importantly, electron diffraction was able to establish that the crystallites were phase pure i.e. no other polymorphic forms were identified throughout the sample. 3D data collected at room temperature circumvented potential phase changes, conveniently preserving the metastable polymorph during structural elucidation. The limited resolution of the electron diffraction data (> 1 Å), combined with the complexity of configurational disorder and possible beam-induced amorphization, meant that the structure could not be obtained by ab-initio direct methods. This is a recurrent situation for nanocrystalline pharmaceutical crystals. Instead, two possible starting models arose from simulated annealing based on diffraction data alone. Density Functional Theory energy minimisation followed, determining the correct model, with an independent validation of the experimental structural solution, comparing favourably to single-crystal X-ray diffraction studies. Our results reveal a promising protocol enabling the exploitation of electron diffraction data with limited resolution obtained from beam sensitive organic materials. The method is widely extendable to a number of pharmaceutical compounds that are not amenable for the growth of large single crystals required by X-ray diffraction, and for which an efficient structure determination is required
Surfactant technology: to new rules, new design required !
No full textWith a growing toolbox of surfactant-mediated chemistry in water, and an increased number of scaled up transformations, came tremendous learning, that we have so far kept undisclosed for the most part .1 These learning opportunities collected in the few expert groups, whether fully understood or still pragmatic, have led to substantial know-how in reaction, process, and synthesis design.2 We would like here to share some of the fundamental principles inherent to micellar catalysis, and illustrate them on a particularly challenging Suzuki-Miyaura cross-coupling case. The structures of the Active Pharmaceutical Ingredient (API) and of its intermediates were blinded for confidentiality reasons but can nevertheless well illustrate important factors that has to be considered (e.g. lipophilicity). Besides, the API taken as an example for the discussion bears high commonality with a lot of other targets with, to name a few in no specific order, the formation of a biphenyl system, an amide, and nucleophilic aromatic substitutions, hence the opportunity to rapidly utilize the learnings in other areas