Vom Lernprozess des Ärztenetzwerks xundart: Innovative und wissenschaftlich fundierte Qualitätsentwicklung

Systemische und reflexive Qualitätsentwicklung ist ein Anliegen des Ostschweizer Ärztenetzwerks xundart. Gerade nicht-quantitative Qualitätsarbeit erfordert von den Mitgliedern einer Organisation Vertrauen, Offenheit zur Selbst- und Fremdbeobachtung und die Bereitschaft, aus den eigenen Komfortzonen herauszutreten. Vonseiten der Verantwortlichen sind sorgfältige Kommunikationsarbeit, Fingerspitzengefühl und Geduld gefragt

Manufacturing considerations for the development of lipid nanoparticles using microfluidics

In the recent of years, the use of lipid nanoparticles (LNPs) for RNA delivery has gained considerable attention, with a large number in the clinical pipeline as vaccine candidates or to treat a wide range of diseases. Microfluidics offers considerable advantages for their manufacture due to its scalability, reproducibility and fast preparation. Thus, in this study, we have evaluated operating and formulation parameters to be considered when developing LNPs. Among them, the flow rate ratio (FRR) and the total flow rate (TFR) have been shown to significantly influence the physicochemical characteristics of the produced particles. In particular, increasing the TFR or increasing the FRR decreased the particle size. The amino lipid choice (cationic—DOTAP and DDAB; ionisable—MC3), buffer choice (citrate buffer pH 6 or TRIS pH 7.4) and type of nucleic acid payload (PolyA, ssDNA or mRNA) have also been shown to have an impact on the characteristics of these LNPs. LNPs were shown to have a high (>90%) loading in all cases and were below 100 nm with a low polydispersity index (≤0.25). The results within this paper could be used as a guide for the development and scalable manufacture of LNP systems using microfluidics

VEGF and the Fab fragment of a humanized neutralizing antibody: crystal structure of the complex at 2.4 å resolution and mutational analysis of the interface

AbstractBackground: Vascular endothelial growth factor (VEGF) is a highly specific angiogenic growth factor; anti-angiogenic treatment through inhibition of receptor activation by VEGF might have important therapeutic applications in diseases such as diabetic retinopathy and cancer. A neutralizing anti-VEGF antibody shown to suppress tumor growth in an in vivo murine model has been used as the basis for production of a humanized version.Results: We present the crystal structure of the complex between VEGF and the Fab fragment of this humanized antibody, as well as a comprehensive alanine-scanning analysis of the contact residues on both sides of the interface. Although the VEGF residues critical for antibody binding are distinct from those important for high-affinity receptor binding, they occupy a common region on VEGF, demonstrating that the neutralizing effect of antibody binding results from steric blocking of VEGF–receptor interactions. Of the residues buried in the VEGF–Fab interface, only a small number are critical for high-affinity binding; the essential VEGF residues interact with those of the Fab fragment, generating a remarkable functional complementarity at the interface.Conclusions: Our findings suggest that the character of antigen–antibody interfaces is similar to that of other protein–protein interfaces, such as ligand–receptor interactions; in the case of VEGF, the principal difference is that the residues essential for binding to the Fab fragment are concentrated in one continuous segment of polypeptide chain, whereas those essential for binding to the receptor are distributed over four different segments and span across the dimer interface

Three-Tiered Risk Stratification Model to Predict Progression in Barrett's Esophagus Using Epigenetic and Clinical Features

Barrett's esophagus predisposes to esophageal adenocarcinoma. However, the value of endoscopic surveillance in Barrett's esophagus has been debated because of the low incidence of esophageal adenocarcinoma in Barrett's esophagus. Moreover, high inter-observer and sampling-dependent variation in the histologic staging of dysplasia make clinical risk assessment problematic. In this study, we developed a 3-tiered risk stratification strategy, based on systematically selected epigenetic and clinical parameters, to improve Barrett's esophagus surveillance efficiency

Using microfluidics for scalable manufacturing of nanomedicines from bench to GMP : a case study using protein-loaded liposomes

Nanomedicines are well recognised for their ability to improve therapeutic outcomes. Yet, due to their complexity, nanomedicines are challenging and costly to produce using traditional manufacturing methods. For nanomedicines to be widely exploited, new manufacturing technologies must be adopted to reduce development costs and provide a consistent product. Within this study, we investigate microfluidic manufacture of nanomedicines. Using protein-loaded liposomes as a case study, we manufacture liposomes with tightly defined physico-chemical attributes (size, PDI, protein loading and release) from small-scale (1 mL) through to GMP volume production (200 mL/min). To achieve this, we investigate two different laminar flow microfluidic cartridge designs (based on a staggered herringbone design and a novel toroidal mixer design); for the first time we demonstrate the use of a new microfluidic cartridge design which delivers seamless scale-up production from bench-scale (12 mL/min) through GMP production requirements of over 20 L/h using the same standardised normal operating parameters. We also outline the application of tangential flow filtration for down-stream processing and high product yield. This work confirms that defined liposome products can be manufactured rapidly and reproducibly using a scale-independent production process, thereby de-risking the journey from bench to approved product

SARS-Coronavirus Replication Is Supported by a Reticulovesicular Network of Modified Endoplasmic Reticulum

Positive-strand RNA viruses, a large group including human pathogens such as SARS-coronavirus (SARS-CoV), replicate in the cytoplasm of infected host cells. Their replication complexes are commonly associated with modified host cell membranes. Membrane structures supporting viral RNA synthesis range from distinct spherular membrane invaginations to more elaborate webs of packed membranes and vesicles. Generally, their ultrastructure, morphogenesis, and exact role in viral replication remain to be defined. Poorly characterized double-membrane vesicles (DMVs) were previously implicated in SARS-CoV RNA synthesis. We have now applied electron tomography of cryofixed infected cells for the three-dimensional imaging of coronavirus-induced membrane alterations at high resolution. Our analysis defines a unique reticulovesicular network of modified endoplasmic reticulum that integrates convoluted membranes, numerous interconnected DMVs (diameter 200–300 nm), and “vesicle packets” apparently arising from DMV merger. The convoluted membranes were most abundantly immunolabeled for viral replicase subunits. However, double-stranded RNA, presumably revealing the site of viral RNA synthesis, mainly localized to the DMV interior. Since we could not discern a connection between DMV interior and cytosol, our analysis raises several questions about the mechanism of DMV formation and the actual site of SARS-CoV RNA synthesis. Our data document the extensive virus-induced reorganization of host cell membranes into a network that is used to organize viral replication and possibly hide replicating RNA from antiviral defense mechanisms. Together with biochemical studies of the viral enzyme complex, our ultrastructural description of this “replication network” will aid to further dissect the early stages of the coronavirus life cycle and its virus-host interactions

A Multicenter, Double-Blinded Validation Study of Methylation Biomarkers for Progression Prediction in Barrett's Esophagus

Esophageal adenocarcinoma risk in Barrett’s esophagus (BE) is increased 30- to 125-fold versus the general population. Among all BE patients, however, neoplastic progression occurs only once per 200 patient-years. Molecular biomarkers are therefore needed to risk-stratify patients for more efficient surveillance endoscopy and to improve the early detection of progression. We therefore performed a retrospective, multicenter, double-blinded validation study of 8 BE progression prediction methylation biomarkers. Progression or nonprogression were determined at 2 years (tier 1) and 4 years (tier 2). Methylation was assayed in 145 nonprogressors (NPs) and 50 progressors (Ps) using real-time quantitative methylation-specific PCR. Ps were significantly older than NPs (70.6 vs. 62.5 years, p < 0.001). We evaluated a linear combination of the 8 markers, using coefficients from a multivariate logistic regression analysis. Areas under the ROC curve (AUCs) were high in the 2-, 4-year and combined data models (0.843, 0.829 and 0.840; p<0.001, p<0.001 and p<0.001, respectively). In addition, even after rigorous overfitting correction, the incremental AUCs contributed by panels based on the 8 markers plus age vs. age alone were substantial (Δ-AUC = 0.152, 0.114 and 0.118, respectively) in all three models. A methylation biomarker-based panel to predict neoplastic progression in BE has potential clinical value in improving both the efficiency of surveillance endoscopy and the early detection of neoplasia

Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment

Dolutegravir twice-daily dosing in children with HIV-associated tuberculosis: a pharmacokinetic and safety study within the open-label, multicentre, randomised, non-inferiority ODYSSEY trial

Background: Children with HIV-associated tuberculosis (TB) have few antiretroviral therapy (ART) options. We aimed to evaluate the safety and pharmacokinetics of dolutegravir twice-daily dosing in children receiving rifampicin for HIV-associated TB. Methods: We nested a two-period, fixed-order pharmacokinetic substudy within the open-label, multicentre, randomised, controlled, non-inferiority ODYSSEY trial at research centres in South Africa, Uganda, and Zimbabwe. Children (aged 4 weeks to <18 years) with HIV-associated TB who were receiving rifampicin and twice-daily dolutegravir were eligible for inclusion. We did a 12-h pharmacokinetic profile on rifampicin and twice-daily dolutegravir and a 24-h profile on once-daily dolutegravir. Geometric mean ratios for trough plasma concentration (Ctrough), area under the plasma concentration time curve from 0 h to 24 h after dosing (AUC0–24 h), and maximum plasma concentration (Cmax) were used to compare dolutegravir concentrations between substudy days. We assessed rifampicin Cmax on the first substudy day. All children within ODYSSEY with HIV-associated TB who received rifampicin and twice-daily dolutegravir were included in the safety analysis. We described adverse events reported from starting twice-daily dolutegravir to 30 days after returning to once-daily dolutegravir. This trial is registered with ClinicalTrials.gov (NCT02259127), EudraCT (2014–002632-14), and the ISRCTN registry (ISRCTN91737921). Findings: Between Sept 20, 2016, and June 28, 2021, 37 children with HIV-associated TB (median age 11·9 years [range 0·4–17·6], 19 [51%] were female and 18 [49%] were male, 36 [97%] in Africa and one [3%] in Thailand) received rifampicin with twice-daily dolutegravir and were included in the safety analysis. 20 (54%) of 37 children enrolled in the pharmacokinetic substudy, 14 of whom contributed at least one evaluable pharmacokinetic curve for dolutegravir, including 12 who had within-participant comparisons. Geometric mean ratios for rifampicin and twice-daily dolutegravir versus once-daily dolutegravir were 1·51 (90% CI 1·08–2·11) for Ctrough, 1·23 (0·99–1·53) for AUC0–24 h, and 0·94 (0·76–1·16) for Cmax. Individual dolutegravir Ctrough concentrations were higher than the 90% effective concentration (ie, 0·32 mg/L) in all children receiving rifampicin and twice-daily dolutegravir. Of 18 children with evaluable rifampicin concentrations, 15 (83%) had a Cmax of less than the optimal target concentration of 8 mg/L. Rifampicin geometric mean Cmax was 5·1 mg/L (coefficient of variation 71%). During a median follow-up of 31 weeks (IQR 30–40), 15 grade 3 or higher adverse events occurred among 11 (30%) of 37 children, ten serious adverse events occurred among eight (22%) children, including two deaths (one tuberculosis-related death, one death due to traumatic injury); no adverse events, including deaths, were considered related to dolutegravir. Interpretation: Twice-daily dolutegravir was shown to be safe and sufficient to overcome the rifampicin enzyme-inducing effect in children, and could provide a practical ART option for children with HIV-associated TB