Extent and Effect of Covering Laryngeal Structures with Synthetic Laryngeal Mucus via Two Different Administration Techniques.

OBJECTIVE: The first goal of this study was to investigate the coverage of laryngeal structures using two potential administration techniques for synthetic mucus: inhalation and lozenge ingestion. As a second research question, the study investigated the potential effects of these techniques on standardized voice assessment parameters. METHODS: Fluorescein was added to throat lozenges and to an inhalation solution to visualize the coverage of laryngeal structures through blue light imaging. The study included 70 vocally healthy subjects. Fifty subjects underwent administration via lozenge ingestion and 20 subjects performed the inhalation process. For the first research question, the recordings from the blue light imaging system were categorized to compare the extent of coverage on individual laryngeal structures objectively. Secondly, a standardized voice evaluation protocol was performed before and after each administration to determine any measurable effects of typical voice parameters. RESULTS: The administration via inhalation demonstrated complete coverage of all laryngeal structures, including the vocal folds, ventricular folds, and arytenoid cartilages, as visualized by the fluorescent dye. In contrast, the application of the lozenge predominantly covered the pharynx and laryngeal surface toward the aryepiglottic fold, but not the inferior structures. All in all, the comparison before and after administration showed no clear effect, although a minor deterioration of the acoustic signal was noted in the shimmer and cepstral peak prominence after the inhalation. CONCLUSIONS: Our findings indicate that the inhalation process is a more effective technique for covering deeper laryngeal structures such as the vocal folds and ventricular folds with synthetic mucus. This knowledge enables further in vivo studies on the role of laryngeal mucus in phonation in general, and how it can be substituted or supplemented for patients with reduced glandular activity as well as for heavy voice users.

3D screen printing technology enables fabrication of oral drug dosage forms with freely tailorable release profiles.

3D printing offers new opportunities to customize oral dosage forms of pharmaceuticals for different patient populations, improving patient safety, care, and compliance. Although several notable 3D print technologies have been developed, such as inkjet printing, powder-based printing, selective laser sintering (SLS) printing, and fused deposition modelling (FDM), among others, their capacity is often limited by the number of printing heads. 3D screen-printing (3DSP) is based on a classic flatbed screen printing that is widely used in industrial applications for technical applications. 3DSP can build up thousands of units per screen simultaneously, enabling mass customization of pharmaceuticals. Here, we use 3DSP to investigate two novel paste formulations: immediate-release (IR) and extended-release (ER) using Paracetamol (acetaminophen) as the active pharmaceutical ingredient (API). Both disk-shaped and donut-shaped tablets were fabricated using one or both pastes to design drug delivery systems (DDS) with tailored API release profiles. The size and mass of the produced tablets demonstrated high uniformity. Characterization of the tablets physical properties, such as breaking force (25-39 N) and friability (0.002-0.237%), adhering to Ph. Eur (10th edition). Finally, drug release tests with a phosphate buffer at pH 5.8 showed Paracetamol release depended on the IR- and ER paste materials and their respective compartment size of the composite DDS, which can be readily varied using 3DSP. This work further demonstrates the potential of 3DSP to manufacture complex oral dosage forms exhibiting custom release functionalities for mass production.

Topical systems for the controlled release of antineoplastic Drugs: Oxidized Alginate-Gelatin Hydrogel/Unilamellar vesicles.

The efficacy of chemotherapeutic procedures relies on delivering proper concentrations of anti-cancer drugs in the tumor surroundings, so as to prevent potential side effects on healthy tissues. Novel drug carrier platforms should not just be able to deliver anticancer molecules, but also allow for adjustements in the way these drugs are administered to the patients. We developed a system for delivering water-insoluble drugs, based on the use of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), or bis(2-ethylhexyl) sulfosuccinate benzyl-n-hexadecyldimethylammonium (BHD-AOT), embedded into oxidized alginate-gelatin (ADA/Gel) hydrogel, emulating a patch for topic applications. After being loaded with curcumin, cancer cells such as human colorectal adenocarcinoma (HCT116 and DLD-1) and melanoma cell lines (MEL501), and non-malignant cells such as mammary epithelial cell lines (NMuMG) and embryonal fibroblasts (NIH 3T3 or NEO cells) were analyzed for biocompatibility and cytotoxic effects. The results show that the proposed system can load comparatively higher concentrations of the drug (with respect to other nano/microcarriers in the literature), and that it can enhance the likelihood of the drug being uptaken by cancer cells instead of non-malignant cells. These assays were complemented by diffusion studies across the stratum corneum of rat skin, with the aim of determining the system's efficiency during topical application. Finally, the stability of the patch was tested after lyophilization to determine its potential pharmaceutical use. As a whole, the combined system represents a highly reliable and robust method for embedding and delivering complex insoluble chemotherapeutical molecules, and it is less invasive than other alternative methods in the literature.

Soluble CD83 improves and accelerates wound healing by the induction of pro-resolving macrophages.

To facilitate the recovery process of chronic and hard-to-heal wounds novel pro-resolving treatment options are urgently needed. We investigated the pro-regenerative properties of soluble CD83 (sCD83) on cutaneous wound healing, where sCD83 accelerated wound healing not only after systemic but also after topical application, which is of high therapeutic interest. Cytokine profile analyses revealed an initial upregulation of inflammatory mediators such as TNFα and IL-1ß, followed by a switch towards pro-resolving factors, including YM-1 and IL-10, both expressed by tissue repair macrophages. These cells are known to mediate resolution of inflammation and stimulate wound healing processes by secretion of growth factors such as epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF), which promote vascularization as well as fibroblast and keratinocyte differentiation. In conclusion, we have found strong wound healing capacities of sCD83 beyond the previously described role in transplantation and autoimmunity. This makes sCD83 a promising candidate for the treatment of chronic- and hard-to-heal wounds.

Skin sodium is increased in male patients with multiple sclerosis and related animal models.

Novel MRI techniques allow a noninvasive quantification of tissue sodium and reveal the skin as a prominent compartment of sodium storage in health and disease. Since multiple sclerosis (MS) immunopathology is initiated in the periphery and increased sodium concentrations induce proinflammatory immune cells, the skin represents a promising compartment linking high sodium concentrations and MS immunopathology. We used a 7-T sodium MRI (23Na-MRI) and inductively coupled plasma mass spectrometry to investigate the skin sodium content in two mouse models of MS. We additionally performed 3-T 23Na-MRI of calf skin and muscles in 29 male relapsing-remitting MS (RRMS) patients and 29 matched healthy controls. Demographic and clinical information was collected from interviews, and disease activity was assessed by expanded disability status scale scoring. 23Na-MRI and chemical analysis demonstrated a significantly increased sodium content in the skin during experimental autoimmune encephalomyelitis independent of active immunization. In male patients with RRMS, 23Na-MRI demonstrated a higher sodium signal in the area of the skin compared to age- and biological sex-matched healthy controls with higher sodium, predicting future disease activity in cranial MRI. In both studies, the sodium enrichment was specific to the skin, as we found no alterations of sodium signals in the muscle or other tissues. Our data add to the recently identified importance of the skin as a storage compartment of sodium and may further represent an important organ for future investigations on salt as a proinflammatory agent driving autoimmune neuroinflammation such as that in MS.

The tangential flow absorption model (TFAM) - A novel dissolution method for evaluating the performance of amorphous solid dispersions of poorly water-soluble actives.

There is a substantial demand for absorptive dissolution tests, as single vessel dissolution experiments were originally not designed for testing supersaturating systems. Current approaches suffer from inadequate mass transfer of the dissolved active from the dissolution site, discrepancies in the fluid volume compared to in vivo intestinal fluid volumes or the dilution of functional excipients. In this work a novel dissolution apparatus was developed that enables adjustable mass transfer of the active through a membrane, while retaining the functional polymeric excipients at the dissolution site. Using this setup the dissolution behavior of various spray dried amorphous solid dispersions containing carbamazepine, hydrochlorothiazide and ketoconazole as model actives at intermediate and high supersaturation levels was evaluated. Compared to non-absorptive dissolution experiments, differences in the concentration-time profiles were noted. The experiments with a high supersaturation of ketoconazole revealed a concentration decrease over time under absorptive conditions. Additionally, it was observed that the difference between "spring" as well as "spring and parachute" formulations was less pronounced with increasing drug efflux. Further, the apparatus was also tested with Fasted State Simulated Intestinal Fluid as dissolution medium and results were compared to phosphate buffer pH6.8. As major benefits of the new TFAM apparatus the easy experimental procedure and sample preparation for drug concentration measurements using spectroscopy in the permeate, without the necessity for additional filtration and/or centrifugation to remove precipitated drug molecules, could be highlighted. This TFAM approach seems to be a promising tool for identifying formulations for amorphous solid dispersions with optimal in vitro performance.

Retronasal olfactory function in patients with smell loss but subjectively normal flavor perception.

OBJECTIVES: The human sense of smell constitutes the main part of flavor perception. Typically, patients with loss of olfactory function complain of diminished perception during eating and drinking. However, some patients with smell loss still report normal enjoyment of foods. The aim of the present study was to compare orthonasal and retronasal olfactory function in patients with non-sinonasal smell loss and subjectively normal flavor perception. METHODS: Nineteen patients (mean age [range] 52.0 [8-83 years]) with self-reported olfactory impairment but subjective normal flavor perception were included. Olfactory performance was assessed using the Sniffin' Sticks (TDI) for orthonasal and the Candy Smell Test (CST) for retronasal function. Visual analogue scales were used for self-assessment of odor (SOP), taste (STP), and flavor perception (SFP), ranging from 0 (no perception) to 10 (excellent perception). RESULTS: Mean (SD) SFP was 8.0 (1.8). Mean (SD) orthonasal TDI-score of all patients was 14.4 (5.3, range 6-25.3) with 11 patients classified as anosmic and eight as hyposmic. Mean/SD retronasal CST-score was 8.8 (2.7, range 3-13) within the range of anosmia/hyposmia. No correlation was found between SFP and the CST (P = .62). CONCLUSION: The present results showed that despite claiming normal flavor perception, our patients were ortho- and retronasally dysosmic using standard tests for olfactory function. Although other explanations could be possible, we suggest that this subjective flavor perception might be due to unconscious memory recall from previously experienced cross-modal sensory interactions. LEVEL OF EVIDENCE: 4 Laryngoscope, 130:1629-1633, 2020.

Improved stability of polyclonal antibodies: A case study with lyophilization-conserved antibodies raised against epitopes from the malaria parasite Plasmodium falciparum.

Antibodies can be produced as polyclonal (pAb) or monoclonal (mAb) liquid formulations with limited shelf-life. For pAbs, unlike mAbs, only little is known about excipients and lyophilization affecting antibody stability upon reconstitution. We used a model pAb directed against Plasmodium falciparum (Pf) pyridoxal 5'-phosphate synthase 2 (Pdx2) to systemically study effects of bulking agents (amino acids, phosphate buffers, salt solutions), sugar(alcohols), surfactants and protein additions (bovine serum albumin, BSA) in liquid pAb formulations (isolated or in combinations) on the activity to detect the antigen in Pf extracts by Western blots. Repeated freeze-thaw cycles (20x) and extended room temperature storage markedly compromised pAb stability, the former being ameliorated by addition of cryoprotectants (glycerol, sucrose). Lyophilization of pure liquid pAb formulation markedly decreased antibody reactivity upon reconstitution which was not preserved by most bulking agents tested (e.g., histidine, arginine, acetate). Among the tested salt solutions (NaCl, Ringer, PBS), phosphate buffered saline had the largest lyoprotective potential, alongside sucrose, but not trehalose or maltitol. Among combinations of excipients, PBS, sucrose, low concentration BSA and Tween potently preserved PfPdx2 stability. Results for PBS were transferable to PfEnolase pAb, indicating that some of the formulations investigated here might be a low-cost solution for more general applicability to pAbs.

Measurement of shrinkage and cracking in lyophilized amorphous cakes. Part IV: Effects of freezing protocol.

The shrinkage and cracking of pure trehalose cakes during lyophilization has been determined quantitatively using different protocols for the freezing step. The influences of shelf cooling rate and of a two-step freezing protocol with holding and annealing phases were investigated. A small change in the shelf cooling rate from 0.4°C to 0.2°C per minute produced surprisingly large increases in shrinkage and reductions in cracking over all trehalose concentrations up to 30% w/v. The two-step freezing protocol also increased shrinkage and reduced cracking in the final-product cakes, especially at trehalose concentrations ≥ 15% and with large vial fill height. A combination of two-step freezing with use of TopLyo vials produced less than 1.5% cracking even at high trehalose concentrations and large fill height. The results give further confirmation of the causal linkage of shrinkage and cracking during lyophilization, and also illustrate how cracking can be greatly reduced by manipulating the freezing protocol.

Measurement of shrinkage and cracking in lyophilized amorphous cakes, part 3: hydrophobic vials and the question of adhesion.

The importance of cake adhesion to the inside vial wall during lyophilization of amorphous trehalose cakes was determined by using hydrophobized vials. The degrees of cake shrinkage and cracking were determined independently by photographic imaging of the cake top surface in a dark cell. Additionally, measurements with microcomputed tomography were performed. Adhesion is found to be a determining factor in both cake shrinkage and cracking. The correlation between cake detachment from the vial inner wall and trehalose concentration indicates that adhesion of the frozen solute phase is a determining factor in shrinkage. The hydrophobized vials give reduced cracking at trehalose concentrations of up to 15%. The reduced wetting of the hydrophobized inside vial wall gives a planar cake topography with a uniform distribution of cracks within the cake.

Measurement of Shrinkage and Cracking in Lyophilized Amorphous Cakes. Part II: Kinetics.

PURPOSE: Measurement of the kinetic development of shrinkage and cracking of an amorphous trehalose cake as they take place during lyophilization. METHODS: A novel technique has been developed which monitors a vial in situ during the freeze-drying cycle. The 2-dimensional degrees of shrinkage and cracking in its top surface are determined quantitatively using a digital camera and evaluated using AxioVision. RESULTS: Shrinkage and cracking develop largely already during programmed primary drying and are coupled. For trehalose, sucrose and maltose no clear correlation between shrinkage and wg' is found. There is no dependence of cake rim detachment from the vial inner surface on the trehalose concentration. Cake adhesion is therefore likely not the only determining factor for detachment and shrinkage. CONCLUSIONS: If shrinkage can occur during primary drying, then this relaxes the drying tension produced by desorption of non-frozen water out of the amorphous structure left behind as the sublimation front passes through a volume element, and causes little or no cracking. If shrinkage is restrained, then the drying tension is relaxed by cracking of the brittle cake.

Measurement of shrinkage and cracking in lyophilized amorphous cakes. Part I: final-product assessment.

An experimental technique is presented to determine independently shrinkage and cracking in lyophilized amorphous cakes based on photographic imaging of their top surface. An inverse correlation between cake shrinkage and cracking during freeze-drying is seen. Shrinkage relaxes the drying tension and gives little cracking, whereas if shrinkage is restrained then more cracking occurs. A lower shrinkage and greater cracking with higher disaccharide concentration correlates with change in cake hardness and brittleness. Adhesion of the cake to the inside vial wall could not be identified as a determining factor for shrinkage. Shrinkage is non-uniform across the cake's surface and is manifested largely in the peripheral region. A correlation between shrinkage and w(g)' for different disaccharides suggests that drying tension develops as non-frozen water is lost from the porous solid after sublimation of the ice phase has exposed the solid/gas interface.

Structural studies of EDTA-induced fibrillation of salmon calcitonin.

PURPOSE: The purpose of this work was to determine the structure of an insoluble precipitate formed when mixing approximately equimolar amounts of ethylenediamine tetraacetic acid (EDTA) and salmon calcitonin (sCT). METHODS: The interaction between EDTA and sCT was examined by measuring solution turbidity kinetics as a function of pH, ionic strength, and addition of ferric ions. Fourier-transformation infrared spectroscopy (FT-IR) identified changes in peptide secondary structure in presence of EDTA. Scanning and transmission electron spectroscopy revealed the macromolecular structure of the sCT/EDTA precipitate. RESULTS: Aggregation of sCT in a time frame up to 1200 min cannot be induced by either pH (range 3.0-7.0) or ionic strength (up to 200 mM) alone, but is a noncovalent interaction between sCT and EDTA. In the pH range 5.0-7.0, a molar binding stoichiometry of sCT/EDTA in the precipitate of 1-3 was determined. We suggest coulombic binding of the free acidic groups of the EDTA to the side chains of the basic amino acids present in the sCT primary sequence. This results in bridging aggregation of the sCT molecules and their precipitation in aqueous solution. The aggregation reaction was blocked by the addition of ferric ions, which bind preferentially to the acidic groups of the EDTA. The sCT/EDTA precipitate redissolves in water in a pH-dependent manner. FT-IR measurements showed a progressive loss of the random coil structure of sCT in water in the presence of EDTA and a simultaneous strong increase in beta-structure. Scanning electron microscopy revealed a fine, sponge-like morphology of the isolated, solid sCT/EDTA precipitate. Transmission electron microscopy delivered final proof of the existence of extensive fibrillation in the sCT/EDTA mixture. CONCLUSIONS: EDTA induces rapid fibrillation of sCT in water and the partially reversible formation of a noncovalent, insoluble sCT/EDTA precipitate.