Chitosan-Based Thermogelling System for Nose-to-Brain Donepezil Delivery: Optimising Formulation Properties and Nasal Deposition Profile.

Donepezil nasal delivery strategies are being continuously investigated for advancing therapy in Alzheimer's disease. The aim of this study was to develop a chitosan-based, donepezil-loaded thermogelling formulation tailored to meet all the requirements for efficient nose-to-brain delivery. A statistical design of the experiments was implemented for the optimisation of the formulation and/or administration parameters, with regard to formulation viscosity, gelling and spray properties, as well as its targeted nasal deposition within the 3D-printed nasal cavity model. The optimised formulation was further characterised in terms of stability, in vitro release, in vitro biocompatibility and permeability (using Calu-3 cells), ex vivo mucoadhesion (using porcine nasal mucosa), and in vivo irritability (using slug mucosal irritation assay). The applied research design resulted in the development of a sprayable donepezil delivery platform characterised by instant gelation at 34 °C and olfactory deposition reaching a remarkably high 71.8% of the applied dose. The optimised formulation showed prolonged drug release (t1/2 about 90 min), mucoadhesive behaviour, and reversible permeation enhancement, with a 20-fold increase in adhesion and a 1.5-fold increase in the apparent permeability coefficient in relation to the corresponding donepezil solution. The slug mucosal irritation assay demonstrated an acceptable irritability profile, indicating its potential for safe nasal delivery. It can be concluded that the developed thermogelling formulation showed great promise as an efficient donepezil brain-targeted delivery system. Furthermore, the formulation is worth investigating in vivo for final feasibility confirmation.

Vitamin D Supplementation and Clinical Outcomes in Severe COVID-19 Patients-Randomized Controlled Trial.

COVID-19 symptoms vary from asymptomatic cases to moderate and severe illness with patients needing hospitalization and intensive care treatment. Vitamin D is associated with severity of viral infections and has an immune-modulatory effect in immune response. Observational studies showed a negative association of low vitamin D levels and COVID-19 severity and mortality outcomes. In this study, we aimed to determine whether daily supplementation of vitamin D during intensive care unit (ICU) stay in COVID-19 patients with severe illness affects clinically relevant outcomes. Patients with COVID-19 disease in need of respiratory support admitted to the ICU were eligible for inclusion. Patients with low vitamin D levels were randomized into one of two groups: the intervention group received daily supplementation of vitamin D and the control group did not receive vitamin D supplementation. In total, 155 patients were randomized: 78 into the intervention group and 77 into the control group. There was no statistically significant difference in number of days spent on respiratory support, although the trial was underpowered for the main outcome. There was no difference in any of the secondary outcomes analyzed between two groups. Our study suggests no benefit in vitamin D supplementation to patients with severe COVID-19 disease admitted to the ICU and in need of respiratory support in any of the analyzed outcomes.

Tailoring functional spray-dried powder platform for efficient donepezil nose-to-brain delivery.

Shortcomings of oral donepezil administration in the treatment of Alzheimer's disease have paved the way for ongoing investigations towards more efficient and safe donepezil nose-to-brain delivery. Herein we present the development of advantageous powder platform for donepezil nose-to-brain delivery, coupling careful design of chitosan and mannitol-based carrier matrix with spray-drying technology advantages and early consideration of adequate nasal administration mode, employing QbD approach. Unprecedentedly, ultrasonic nozzle was used to atomise the drying feed in response to size-related requirements for nasal aerosol particles. The optimised spray-drying process resulted in free-flowable dry powder with a great majority of particles larger than 10 µm, ensuring localised nasal deposition upon aerosolization, as evidenced by using 3D-printed nasal cavity model. QbD approach coupling formulation, process and administration parameters enabled optimisation of drug deposition profile reaching tremendously high 65.5 % of the applied dose deposited in the olfactory region. The leading formulation exhibited favourable swelling, mucoadhesion, drug release and permeation-enhancing properties, suiting the needs for efficient brain-targeted delivery. Results of in vitro biocompatibility and physico-chemical stability studies confirmed the leading formulation potential for safe and efficient donepezil nose-to-brain delivery. The obtained results encourage extending the study to an appropriate in vivo model needed for the final proof-of-concept.

In situ gelling nanosuspension as an advanced platform for fluticasone propionate nasal delivery.

In this work we present the development of in situ gelling nanosuspension as advanced form for fluticasone propionate nasal delivery. Drug nanocrystals were prepared by wet milling technique. Incorporation of drug nanocrystals into polymeric in situ gelling system with pectin and sodium hyaluronate as constitutive polymers was fine-tuned attaining appropriate formulation surface tension, viscosity and gelling ability. Drug nanonisation improved the release profile and enhanced formulation mucoadhesive properties. QbD approach combining formulation and administration parameters resulted in optimised nasal deposition profile, with 51.8% of the dose deposited in the middle meatus, the critical region in the treatment of rhinosinusitis and nasal polyposis. Results obtained in biocompatibility and physico-chemical stability studies confirmed the leading formulation potential for safe and efficient nasal corticosteroid delivery.

Significant Associations of IgG Glycan Structures With Chronic Graft-Versus-Host Disease Manifestations: Results of the Cross-Sectional NIH Cohort Study.

Chronic graft-versus-host disease (cGvHD) is a systemic alloimmune and autoimmune disorder and a major late complication of allogeneic hematopoietic stem cell transplantation (alloHSCT). The disease is characterized by an altered homeostasis of the humoral immune response. Immunoglobulin G (IgG) glycoprotein is the main effector molecule of the humoral immune response. Changes in IgG glycosylation are associated with a number of autoimmune diseases. IgG glycosylation analysis was done by the means of liquid chromatography in the National Institutes of Health (NIH) cohort of 213 cGvHD patients. The results showed statistically significant differences with regards to cGvHD NIH joint/fascia and skin score, disease activity and intensity of systemic immunosuppression. ROC analysis confirmed that IgG glycosylation increases specificity and sensitivity of models using laboratory parameters and markers of inflammation associated with cGvHD (eosinophil count, complement components C3 and C4 and inflammation markers: albumin, CRP and thrombocyte count). This research shows that IgG glycosylation may play a significant role in cGvHD pathology. Further research could contribute to the understanding of the disease biology and lead to the clinical biomarker development to allow personalized approaches to chronic GvHD therapy.

A Dry Powder Platform for Nose-to-Brain Delivery of Dexamethasone: Formulation Development and Nasal Deposition Studies.

Nasal route of administration offers a unique opportunity of brain targeted drug delivery via olfactory and trigeminal pathway, providing effective CNS concentrations at lower doses and lower risk for adverse reactions compared to systemic drug administration. Therefore, it has been recently proposed as a route of choice for glucocorticoids to control neuroinflammation processes in patients with severe Covid-19. However, appropriate delivery systems tailored to enhance their efficacy yet need to emerge. In this work we present the development of sprayable brain targeting powder delivery platform of dexamethasone sodium phosphate (DSP). DSP-loaded microspheres, optimised employing Quality-by-Design approach, were blended with soluble inert carriers (mannitol or lactose monohydrate). Powder blends were characterized in terms of homogeneity, flow properties, sprayability, in vitro biocompatibility, permeability and mucoadhesion. Nasal deposition studies were performed using 3D printed nasal cavity model. Mannitol provided better powder blend flow properties compared to lactose. Microspheres blended with mannitol retained or enlarged their mucoadhesive properties and enhanced DSP permeability across epithelial model barrier. DSP dose fraction deposited in the olfactory region reached 17.0% revealing the potential of developed powder platform for targeted olfactory delivery. The observed impact of nasal cavity asymmetry highlighted the importance of individual approach when aiming olfactory region.

Glycosylation of immunoglobulin G is regulated by a large network of genes pleiotropic with inflammatory diseases.

Effector functions of immunoglobulin G (IgG) are regulated by the composition of a glycan moiety, thus affecting activity of the immune system. Aberrant glycosylation of IgG has been observed in many diseases, but little is understood about the underlying mechanisms. We performed a genome-wide association study of IgG N-glycosylation (N = 8090) and, using a data-driven network approach, suggested how associated loci form a functional network. We confirmed in vitro that knockdown of IKZF1 decreases the expression of fucosyltransferase FUT8, resulting in increased levels of fucosylated glycans, and suggest that RUNX1 and RUNX3, together with SMARCB1, regulate expression of glycosyltransferase MGAT3. We also show that variants affecting the expression of genes involved in the regulation of glycoenzymes colocalize with variants affecting risk for inflammatory diseases. This study provides new evidence that variation in key transcription factors coupled with regulatory variation in glycogenes modifies IgG glycosylation and has influence on inflammatory diseases.

Choosing proper normalization is essential for discovery of sparse glycan biomarkers.

Rapid progress in high-throughput glycomics analysis enables the researchers to conduct large sample studies. Typically, the between-subject differences in total abundance of raw glycomics data are very large, and it is necessary to reduce the differences, making measurements comparable across samples. Essentially there are two ways to approach this issue: row-wise and column-wise normalization. In glycomics, the differences per subject are usually forced to be exactly zero, by scaling each sample having the sum of all glycan intensities equal to 100%. This total area (row-wise) normalization (TA) results in so-called compositional data, rendering many standard multivariate statistical methods inappropriate or inapplicable. Ignoring the compositional nature of the data, moreover, may lead to spurious results. Alternatively, a log-transformation to the raw data can be performed prior to column-wise normalization and implementing standard statistical tools. Until now, there is no clear consensus on the appropriate normalization method applied to glycomics data. Nor is systematic investigation of impact of TA on downstream analysis available to justify the choice of TA. Our motivation lies in efficient variable selection to identify glycan biomarkers with regard to accurate prediction as well as interpretability of the model chosen. Via extensive simulations we investigate how different normalization methods affect the performance of variable selection, and compare their performance. We also address the effect of various types of measurement error in glycans: additive, multiplicative and two-component error. We show that when sample-wise differences are not large row-wise normalization (like TA) can have deleterious effects on variable selection and prediction.

Decreased Immunoglobulin G Core Fucosylation, A Player in Antibody-dependent Cell-mediated Cytotoxicity, is Associated with Autoimmune Thyroid Diseases.

Autoimmune thyroid diseases (AITD) are the most common group of autoimmune diseases, associated with lymphocyte infiltration and the production of thyroid autoantibodies, like thyroid peroxidase antibodies (TPOAb), in the thyroid gland. Immunoglobulins and cell-surface receptors are glycoproteins with distinctive glycosylation patterns that play a structural role in maintaining and modulating their functions. We investigated associations of total circulating IgG and peripheral blood mononuclear cells glycosylation with AITD and the influence of genetic background in a case-control study with several independent cohorts and over 3,000 individuals in total. The study revealed an inverse association of IgG core fucosylation with TPOAb and AITD, as well as decreased peripheral blood mononuclear cells antennary α1,2 fucosylation in AITD, but no shared genetic variance between AITD and glycosylation. These data suggest that the decreased level of IgG core fucosylation is a risk factor for AITD that promotes antibody-dependent cell-mediated cytotoxicity previously associated with TPOAb levels.

Effects of Environmental Factors on Severity and Mortality of COVID-19.

Background: Most respiratory viruses show pronounced seasonality, but for SARS-CoV-2, this still needs to be documented. Methods: We examined the disease progression of COVID-19 in 6,914 patients admitted to hospitals in Europe and China. In addition, we evaluated progress of disease symptoms in 37,187 individuals reporting symptoms into the COVID Symptom Study application. Findings: Meta-analysis of the mortality risk in seven European hospitals estimated odds ratios per 1-day increase in the admission date to be 0.981 (0.973-0.988, p < 0.001) and per increase in ambient temperature of 1°C to be 0.854 (0.773-0.944, p = 0.007). Statistically significant decreases of comparable magnitude in median hospital stay, probability of transfer to the intensive care unit, and need for mechanical ventilation were also observed in most, but not all hospitals. The analysis of individually reported symptoms of 37,187 individuals in the UK also showed the decrease in symptom duration and disease severity with time. Interpretation: Severity of COVID-19 in Europe decreased significantly between March and May and the seasonality of COVID-19 is the most likely explanation.

Heritability of Human Plasma N-Glycome.

The N-glycosylation profile of total human plasma proteins could be a useful biomarker for various pathological states. Reliable high-throughput methods for such profiling have been developed. However, studies of relative importance of genetic and environmental factors in regulating plasma N-glycome are scarce. The aim of our study was to determine the role of genetic factors in phenotypic variation of plasma N-glycan profile through the estimates of its heritability. Thirty-nine total plasma N-glycome traits were analyzed in 2816 individuals from the TwinsUK data set. For the majority of the traits, high heritability estimates (>50%) were obtained pointing at a significant contribution of genetic factors in plasma N-glycome variation, especially for glycans mostly attached to immunoglobulins. We have also found several structures with higher environmental contribution to their variation.

Fine-Mapping of the Human Blood Plasma N-Glycome onto Its Proteome.

Most human proteins are glycosylated. Attachment of complex oligosaccharides to the polypeptide part of these proteins is an integral part of their structure and function and plays a central role in many complex disorders. One approach towards deciphering this human glycan code is to study natural variation in experimentally well characterized samples and cohorts. High-throughput capable large-scale methods that allow for the comprehensive determination of blood circulating proteins and their glycans have been recently developed, but so far, no study has investigated the link between both traits. Here we map for the first time the blood plasma proteome to its matching N-glycome by correlating the levels of 1116 blood circulating proteins with 113 N-glycan traits, determined in 344 samples from individuals of Arab, South-Asian, and Filipino descent, and then replicate our findings in 46 subjects of European ancestry. We report protein-specific N-glycosylation patterns, including a correlation of core fucosylated structures with immunoglobulin G (IgG) levels, and of trisialylated, trigalactosylated, and triantennary structures with heparin cofactor 2 (SERPIND2). Our study reveals a detailed picture of protein N-glycosylation and suggests new avenues for the investigation of its role and function in the associated complex disorders.

Innovative sprayable in situ gelling fluticasone suspension: Development and optimization of nasal deposition.

The aim of this study was to develop an innovative in situ gelling suspension for effective nasal delivery of fluticasone. Pectin, gellan gum and sodium hyaluronate were used as gelling/thickening agents, and Tween 80 as a suspending agent. The influence of the formulation and/or administration parameters on formulation sprayability and nasal deposition was explored with an appropriate experimental design with the range for parameters in the design obtained from previous research and domain knowledge. All formulations exhibited appropriate sprayability and instant gelation upon mixing with simulated nasal fluid exhibiting weak gel properties convenient for nasal delivery. Targeted turbinate deposition depended on administration and formulation parameters, including their interactions. Decrease in the administration angle from horizontal plane, increase in inspiratory flow and presence of sodium hyaluronate significantly increased deposition in turbinate region. Parameters in interactions included concentration of polymers, surfactant and fluticasone, as well as administration angle. Selected formulations with high turbinate deposition exhibited significant increase in viscosity upon gelation, showing potential to prolong the drug retention at the nasal mucosa. The highest effect on the gel viscosity, strength and fluticasone release profile was observed for gellan gum, thus recognised as crucial parameter for the optimisation of overall therapeutic effect.

Increased central adiposity is associated with pro-inflammatory immunoglobulin G N-glycans.

BACKGROUND: Increased body fat may be associated with an increased risk of developing an underlying pro-inflammatory state, thus leading to greater risk of developing certain chronic conditions. Immunoglobulin G has the ability to exert both anti- and pro-inflammatory effects, and the N-glycosylation of the fragment crystallisable portion is involved in mediating this process. Body mass index, a rudimentary yet gold standard indication for body fat, has been shown to be associated with agalactosylated immunoglobulin G N-glycans. AIM: We aimed to determine the association between increased body fat and the immunoglobulin G glycosylation features, comparing body mass index to other measures of body fat distribution. METHODS: We investigated a sample of 637 community-based 45-69 year olds, with mixed phenotypes, residing in Busselton, Western Australia. Body mass index and the waist-to-hip and waist-to-height ratios were calculated using anthropometry, while dual-energy x-ray absorptiometry was performed to gain an accurate measure of total and area specific body fat. Serum immunoglobulin GN-glycans were analysed by ultra-performance liquid chromatography. RESULTS: Twenty-two N-glycan peaks were found to be associated with at least one of the fat measures. While the previous association of body mass index to agalactosylated immunoglobulin G was replicated, measures of central adiposity explained the most variation in the immunoglobulin G glycome. CONCLUSION: Central adiposity is associated with an increased pro-inflammatory fraction of immunoglobulin G, suggesting that the android/gynoid ratio or waist-to-height ratio instead be considered when controlling for adiposity in immunoglobulin G glycome biomarker studies.

Publisher Correction: Network inference from glycoproteomics data reveals new reactions in the IgG glycosylation pathway.

Correction to: Nature Communications (2017) 8:1231. doi:10.1038/s41467-017-01525-0.

Unravelling Immunoglobulin G Fc N-Glycosylation: A Dynamic Marker Potentiating Predictive, Preventive and Personalised Medicine.

Multiple factors influence immunoglobulin G glycosylation, which in turn affect the glycoproteins' function on eliciting an anti-inflammatory or pro-inflammatory response. It is prudent to underscore these processes when considering the use of immunoglobulin G N-glycan moieties as an indication of disease presence, progress, or response to therapeutics. It has been demonstrated that the altered expression of genes that encode enzymes involved in the biosynthesis of immunoglobulin G N-glycans, receptors, or complement factors may significantly modify immunoglobulin G effector response, which is important for regulating the immune system. The immunoglobulin G N-glycome is highly heterogenous; however, it is considered an interphenotype of disease (a link between genetic predisposition and environmental exposure) and so has the potential to be used as a dynamic biomarker from the perspective of predictive, preventive, and personalised medicine. Undoubtedly, a deeper understanding of how the multiple factors interact with each other to alter immunoglobulin G glycosylation is crucial. Herein we review the current literature on immunoglobulin G glycoprotein structure, immunoglobulin G Fc glycosylation, associated receptors, and complement factors, the downstream effector functions, and the factors associated with the heterogeneity of immunoglobulin G glycosylation.

IgG glycosylation and DNA methylation are interconnected with smoking.

BACKGROUND: Glycosylation is one of the most common post-translation modifications with large influences on protein structure and function. The effector function of immunoglobulin G (IgG) alters between pro- and anti-inflammatory, based on its glycosylation. IgG glycan synthesis is highly complex and dynamic. METHODS: With the use of two different analytical methods for assessing IgG glycosylation, we aim to elucidate the link between DNA methylation and glycosylation of IgG by means of epigenome-wide association studies. In total, 3000 individuals from 4 cohorts were analyzed. RESULTS: The overlap of the results from the two glycan measurement panels yielded DNA methylation of 7 CpG-sites on 5 genomic locations to be associated with IgG glycosylation: cg25189904 (chr.1, GNG12); cg05951221, cg21566642 and cg01940273 (chr.2, ALPPL2); cg05575921 (chr.5, AHRR); cg06126421 (6p21.33); and cg03636183 (chr.19, F2RL3). Mediation analyses with respect to smoking revealed that the effect of smoking on IgG glycosylation may be at least partially mediated via DNA methylation levels at these 7 CpG-sites. CONCLUSION: Our results suggest the presence of an indirect link between DNA methylation and IgG glycosylation that may in part capture environmental exposures. GENERAL SIGNIFICANCE: An epigenome-wide analysis conducted in four population-based cohorts revealed an association between DNA methylation and IgG glycosylation patterns. Presumably, DNA methylation mediates the effect of smoking on IgG glycosylation.

Network inference from glycoproteomics data reveals new reactions in the IgG glycosylation pathway.

Immunoglobulin G (IgG) is a major effector molecule of the human immune response, and aberrations in IgG glycosylation are linked to various diseases. However, the molecular mechanisms underlying protein glycosylation are still poorly understood. We present a data-driven approach to infer reactions in the IgG glycosylation pathway using large-scale mass-spectrometry measurements. Gaussian graphical models are used to construct association networks from four cohorts. We find that glycan pairs with high partial correlations represent enzymatic reactions in the known glycosylation pathway, and then predict new biochemical reactions using a rule-based approach. Validation is performed using data from a GWAS and results from three in vitro experiments. We show that one predicted reaction is enzymatically feasible and that one rejected reaction does not occur in vitro. Moreover, in contrast to previous knowledge, enzymes involved in our predictions colocalize in the Golgi of two cell lines, further confirming the in silico predictions.

Laboratory Experimental Design for a Glycomic Study.

Proper attention to study design before, careful conduct of procedures during, and appropriate inference from results after scientific experiments are important in all scientific studies in order to ensure valid and sometimes definitive conclusions can be made. The design of experiments, also called experimental design, addresses the challenge of structuring and conducting experiments to answer the questions of interest as clearly and efficiently as possible.